Source: NORTHERN ARIZONA UNIVERSITY submitted to
PREVENTING BOVINE BABESIOSIS BY CHARACTERIZING CATTLE FEVER TICK ACARICIDE RESISTANCE MECHANISMS AND GENETIC VARIATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1015605
Grant No.
2018-67015-28301
Project No.
ARZW-2017-05745
Proposal No.
2017-05745
Multistate No.
(N/A)
Program Code
A1221
Project Start Date
Jul 1, 2018
Project End Date
Jun 30, 2022
Grant Year
2018
Project Director
Busch, J. D.
Recipient Organization
NORTHERN ARIZONA UNIVERSITY
(N/A)
FLAGSTAFF,AZ 86011
Performing Department
The Pathogen & Micro Institute
Non Technical Summary
Cattle fever (bovine babesiosis) is a disease that is especially lethal in adult cattle that have not been previously exposed to it. It is caused by several different species of parasites but spread only by cattle fever ticks, which are invasive in the US and Mexico. Cattle fever was once widespread throughout the southern US and caused annual losses to the cattle industry that would amount to more than $3 billion/yr today. Fortunately, intensive efforts by federal and state agencies led to the eradication of cattle fever in almost all of the US by 1960. The successful eradication of the disease was accomplished by using chemicals to eradicate cattle fever ticks. However, the US now imports about 1 million cattle each year from Mexico, where cattle fever ticks and the parasites remain rampant. To make matters worse, tick resistance to chemicals has greatly increased in Mexico since the 1980s and is spreading into the US. Despite rigorous measures by the USDA-APHIS to prevent Mexican cattle from carrying ticks into the US, tick infestations have increased in southern Texas and are spreading north and east. This could lead to the reintroduction of cattle fever into the southern US, which would be devastating to the US cattle industry.Because chemical control is becoming less effective on resistant ticks, an alternative strategy for tick control is to vaccinate cattle against the ticks themselves. When ticks attach and begin to feed, a vaccinated cow will have immune system defenses (antibodies) circulating in its blood that will attack the tick as it feeds. The blood of immunized cattle causes significant mortality in ticks and greatly reduces their reproduction. However, current anti-tick vaccines are not always effective against all cattle fever ticks, probably because a large amount of genetic variation is present in tick populations. The effectiveness of these vaccines can be improved by characterizing the genetic variation present in tick genes used as vaccine candidates, and developing more diverse vaccines that are broadly effective against all cattle fever tick populations. Because anti-tick vaccines and chemicals are the two primary management tools used to prevent cattle fever ticks and their diseases from entering the US, this project will provide critically needed information to our APHIS collaborators who depend on these management tools. We will use genetic analysis to improve tick control by 1) characterizing genetic mechanisms of resistance to chemicals used for tick control, and 2) identifying genetic variation in tick genes that are high priority candidates as anti-tick vaccine targets. In doing so, this project will secure the sustainability and competiveness of the US cattle industry by preventing cattle fever from becoming reestablished in the US.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31233101080100%
Knowledge Area
312 - External Parasites and Pests of Animals;

Subject Of Investigation
3310 - Beef cattle, live animal;

Field Of Science
1080 - Genetics;
Goals / Objectives
The specific science objectives of this project are:Objective 1) Identify and characterize new mechanisms of resistance in R. microplus to four acaricides used for tick control.Objective 2) Identify nucleotide variation in 15 genes encoding tick antigens that are candidates for vaccine targets.Cattle fever ticks (Rhipicephalus microplus and R. annulatus) and the pathogens they transmit are one of the greatest detriments to the livestock industry worldwide. The tick-borne Babesia complex leads to large cumulative losses due to mortality and reduced productivity and requires significant financial investments in the form of treatment of disease and tick control. The overall, long-term goal of this project is to secure the sustainability and competiveness of the US cattle industry by developing genetic tools to prevent cattle fever (bovine babesiosis) from becoming reestablished in the US. Historically, cattle fever was one of the most problematic diseases impacting the US cattle industry but it was successfully eliminated by eradicating the tick vectors; this strategy remains the single most effective means of preventing cattle fever. However, the disease remains endemic in Mexico and its reintroduction into the US is only prevented by the rigorous enforcement of a tick quarantine area in Texas. Unfortunately, the quarantine line has been breached repeatedly in recent years, and tick populations are moving northward, far into southern Texas. We will address this problem by providing information that will lead to a significant improvement in tools used to control cattle fever ticks and prevent cattle fever. We have two primary objectives:Characterize new genetic mechanisms of resistance for commonly used acaricide chemicals; andIdentify genetic variation in tick genes coding for antigens that are high priority candidates as targets for current and future anti-tick vaccines.These objectives will provide the Cattle Fever Tick Eradication Program (CFTEP) with critically needed information about the two leading management tools that can be used to eradicate cattle fever ticks in southern Texas and decrease the risk of Babesia reintroduction: acaricides and vaccines. For Objective #1, we will use existing laboratory tick colonies maintained by the USDA at the Cattle Fever Tick Research Laboratory (CFTRL) in Edinburg, TX. The resistant and susceptible lineages of R. microplus maintained at this facility are highly valuable resources that will refine our understanding of resistance mechanisms in cattle fever ticks. In Objective #2, we will define genetic variation at a set of 15 genes that are the focus of current and/or future vaccine efforts. To do this we will use existing tick collections from a wide geographic range, including Texas, Mexico, Puerto Rico, and Brazil and seek additional collections from India, Africa and Australia. A deeper understanding of tick genetic variation at specific target genes will be highly valuable for current and future development of anti-tick vaccines. The findings of both objectives will allow the USDA to deploy limited resources for tick and disease prevention and control in the most effective manner possible by providing new molecular targets for surveillance. This will allow more effective control of cattle fever tick vectors and the Babesia parasites they carry through improved use of acaricides and vaccines.
Project Methods
Objective #1: Discovery of target site SNPs. We will use an amplicon sequencing (AmpSeq) approach to sequence important target sites in genes known to be important for acaricide resistance. We will target up to 10 genes, with an average of 5 PCR amplicons needed to cover the most important exons of each gene (50 amplicons total per tick). Using the publically available genome for Rhipicephalus microplus, we will design 5 primer pairs for each gene, then amplify all targets in a small number of multiplexed PCRs. All amplicons can be pooled before sequencing on a MiSeq instrument. We will perform this AmpSeq procedure on a set of 300 diverse ticks that include acaricide resistant and susceptible individuals (see paragraph on "Tick Samples" below). A single MiSeq sequencing run will yield hundreds to thousands of reads for all 50 amplicons in all 300 ticks. New SNPs will be validated by re-running AmpSeq or performing Sanger sequencing on a subset of mutant individuals.Tick Samples: We will use resistant and susceptible tick collections to identify SNPs correlated with resistance using AmpSeq. Our target is to analyze 300 ticks that have already been sampled from geographically diverse areas in Texas, Mexico, Puerto Rico, and Brazil. We plan to include ticks from other regions such as India, Australia, and Africa by reaching out to our global network of tick collaborators. The AmpSeq procedure uses DNA as starting template and this gives us great flexibility when choosing tick samples for this study.Objective #1: Discovery of metabolic detoxification mechanisms with RNAseq. A comparison of differential gene expression has not been used previously in R. microplus to characterize broad transcriptomic differences between resistant and susceptible strains. Therefore, we will use total RNA sequencing (RNAseq) on ticks challenged in bioassays with acaricide chemicals to search for transcriptomic differences among resistant and susceptible ticks. Because of their small size, larval ticks yield only small amounts of RNA (<5ng). To increase RNA yield, we are prepared to use pools of tick larvae (20-50 ticks per pool) from the resistant and susceptible ticks exposed to each individual acaricide. We will sequence RNA from pools of resistant and susceptible larvae (6 pools each) sampled from 5 bioassays (permethrin will be included as a control mechanism) and susceptible control larvae (2 lineages) for a total of 72 RNAseq runs. These numbers are small enough that we can re-run the RNAseq step on additional larval pool replicates if needed. Libraries will be run on a NextSeq instrument owned by the PMI research group. PMI has developed an in-house bioinformatics pipeline for transcriptome analysis, in which sequence reads are aligned to a reference genome using BWA-MEM. Read counts are assigned to coding sequences in a Python framework, HTSeq-count. Differential expression is then identified among treatments in DESeq software, using a p-value for significance that is adjusted for false detection rate; transcripts are de novo assembled with metaSPADes. A useful model of constitutive gene expression in R. microplus is elongation factor 1-alpha (ELF1a), which we will use to establish baseline levels of expression in larvae. We will search for broad transcriptomic differences among resistant and susceptible larval pools by setting high thresholds of differential expression (at least 5-10 fold or greater). We will also use a targeted analysis to focus on expression in "classic" genes involved with metabolic resistance (i.e., P450s, carboxylesterases, GSTs, and ABCts). Finally, transcriptome data will allow us to identify new SNPs that may exist between resistant and susceptible ticks at targeted loci.Follow-up analysis: The final outcome of our AmpSeq and RNAseq analysis in Objective #1 will be a prioritized a list of SNPs and candidate genes correlated with resistance. A logical follow-up analysis will be protein modeling to predict if these SNP will have an effect on protein conformation. The most promising SNPs (highly correlated with resistance) will be incorporated into a targeted AmpSeq assay that will be used as a genetic tool to screen field tick collections. We will validate this AmpSeq assay on laboratory and field tick collections that have been screened in bioassays. This tool will provide a valuable method for identifying tick populations that may be acaricide resistant and worth investigating with bioassays.Objective #2: Evaluate genetic variation at candidate genes for anti-tick vaccines. We will generate DNA sequences for targeted exons of each candidate gene using AmpSeq. This will allow us to use DNA as template, which facilitates the use of the diverse tick samples already in our collection. Many exons can be sequenced simultaneously at 300 ticks because the AmpSeq approach is highly multiplexible (described above) and PCR targets can be designed using the gene-enriched R. microplus genome. Sequences will be analyzed with software designed by PMI specifically for the analysis of AmpSeq reads. We will use sequence alignments to document the variable and conserved regions of each exon target.Objective #2: Biological testing of tick protective antigens. We will perform a pilot test of the most promising candidates (5-10 genes) to determine their ability to raise an antibody response in a laboratory mammalian model (rabbit). Synthetic peptides will be designed based on DNA sequence conservation and the proteomic selection criteria above. Target genes may have one or more useful peptides; for instance, past research on aquaporin BmAQP-2 used 3 peptides for stimulating an antibody response. Synthetic peptides will be purchased commerically and shipped to a diagnostic lab to produce rabbit polyclonal antibodies. Polyclonal sera will be tested for the ability to recognize full-length (native) protein on a western blot and then tested for its effect on larval ticks using a membrane feeding system. Dr. Scoles' laboratory at WSU has successfully fed larval R. microplus to repletion on serum using this system. The end result will be a prioritized list of candidate peptides to test in cattle (a future grant proposal) that could eventually serve as globally protective anti-tick vaccines.

Progress 07/01/18 to 06/30/22

Outputs
(N/A)

Impacts
What was accomplished under these goals? OVERALL IMPACTS The highly invasive southern cattle tick (Rhipicephalus microplus) has spread to many regions of the world and transmits a parasite that causes bovine babesiosis, a lethal disease of cattle. This disease and tick occur throughout Mexico and present a constant risk of introduction into Texas - the top beef producing state in the US. Because babesiosis can only be transmitted by R. microplus, the US cattle fever tick eradication control program (CFTEP) manages babesiosis by eliminating tick populations in southern Texas with acaricide chemicals; however, we are in danger of losing this management tool because resistance to all six major classes of acaricides has evolved in Mexico and resistant tick populations are spilling into the US. Our project has led to a change in knowledge concerning this invasive disease system by 1) increasing the understanding of resistance mechanisms in R. microplus and 2) identifying specific improvements to anti-tick vaccines. One immediate impact for tick management emerging from our study is that 60-85% of new tick infestations in Texas carry mutations for resistance to synthetic pyrethroid (SP) acaricides like permethrin, doubling the number of 10 years ago. This means Texas ranchers should no longer use SPs to treat infested cattle, even though they are widely available and affordable. The novel information we have generated across this project carries a high potential for supporting changes in condition for the babesiosis and tick problem through improvements to management tools aimed at securing the long-term productivity of US beef producers. Goal #1. Characterize new genetic mechanisms of resistance for commonly used acaricide chemicals (100% complete) In this goal we generated mRNA sequences from the larvae of four resistant tick colonies maintained at the CFTRL lab in Edinburg, TX. A fifth colony (Deutsch) was included as a susceptible lineage for comparison; this same colony was used for the first whole genome sequence of R. microplus. We used these five colonies to generate large transcriptome datasets that are a foundational resource for ongoing bioinformatic exploration. We have identified many differentially expressed (DE) loci in resistant ticks that will be the focus of our future validation studies. 1) Coumaphos: 26 genes were highly DE in resistant larval pools compared to susceptible pools. Most loci were hypothetical proteins but one highly DE locus was a neuro-peptide protein, a type of small signaling molecule responsible for long-lasting modulation of synaptic transmission. This might point to a mechanism that compensates for the effect of coumaphos when it binds to acetylcholinesterase (AChE). We also looked at expression in many AChE genes, because previous work by our team member, Dr. Pia Olafson, has shown that certain alleles within this gene family are associated with resistance in R. microplus. We found five moderately DE AChE loci, which could suggest upregulation of specific AChE genes might be important. 2) Amitraz: We identified 18 genes that were highly DE in resistant larval pools. The top locus was a dehydrogenase/reductase that could play a role in metabolic detoxification. Five cytochrome-P450 loci were moderately DE, three of which appear to be inducible - larvae exposed to a low level of amitraz were slightly upregulated (1.5 to 2-fold), while those exposed to a high level were strongly upregulated (3-fold and higher). This is a significant finding because other P450 genes have been associated with amitraz resistance in R. microplus. 3) Fipronil: 41 genes were highly DE in resistant larval pools compared. Of the top five, three were structural proteins associated with keratin and cuticular proteins, which could be important because thicker exoskeletons are a known mechanism of chemical resistance. We found four AChE loci that were DE in resistant larvae and looked closely at expression of the GABA receptor, a neuron chloride channel targeted by fipronil. We found that none of the four GABA receptor loci (two alpha and two beta copies) were DE. However, mRNA sequences from the resistant strain (Santa Luiza) were all homozygous for resistance mutation T290N in one of the alpha-GABA genes. 4) Ivermectin: 74 genes were highly DE in resistant larval pools. Most loci were hypothetical proteins that will require extensive investigation before their role in resistance to macrocyclic lactones is understood. One P450 locus was highly upregulated, and another 16 showed only moderate upregulation in resistant larvae. Other markers thought to be associated with ivermectin resistance (ATP-binding transporters, GSTs, and glutamate-gated chlorine channels) did not show significant DE. Another impact from Goal 1 is that we identified new DNA mutations possibly associated with resistance to 3 types of acaricides (SPs, amitraz, and fipronil). We did so by screening 4 target genes using AmpSeq, a new genomics tool that can rapidly screen hundreds of samples simultaneously (see Products section of this report). After running >1,900 ticks from Mexico (16 states) and >1,000 from Texas, we identified two new low-frequency mutations (M918T in domain II and F1538V in domain III) in the VGSC gene, which is known to be associated with resistance. This brings the total number of known R. microplus mutations to five. We have been tracking the spread of each SP mutation from Mexico, found that >60% of new tick infestations in Texas carry resistance mutations for SPs. Two recent publications resulted from our work on SP mutations (Klafke et al. 2019, Thomas et al. 2020). For amitraz resistance, we found two known mutations (T8P and L22S) in the Oct/Tyr receptor (Chen et al. 2007) in Mexico, plus 10 new mutations, one of which occurs in transmembrane region 1 (TM1). Likewise, we found two known mutations (T290N and G337E) in the beta-GABA gene (Hope et al. 2010) known to be associated with fipronil resistance, plus three new mutations in TM2. Overall, we have uncovered new information on acaracide mutations that will improve integrated pest management strategies for controlling cattle fever ticks in the US. Goal #2: Identify genetic variation in tick genes coding for antigens that are high priority candidates as targets for future anti-tick vaccines. (100% complete) We analyzed genetic variation at 13 genes in 92 R. microplus samples from North and South America, and discovered a broad gradient in the number of amino acid substitutions per protein. We ranked genes into three major categories of diversity: Low (0-0.02): voltage-dependent anion channel (VDAC), serpin-1, aquaporin-1 (AQP1), and subolesin Medium (0.02-0.05): AQP2, COIII, GST, and chitinase High (0.05-0.015): serpin-11, voraxin, serpin-5, Bm86, and reprolysin Genes in the low diversity category are very conserved, and based solely on this consideration we suggest they could be high priority vaccine candidates. However, we note that each of the 13 genes contained short segments of conserved sequence, which could still serve as useful targets for future vaccines. These data comprise a previously unavailable dataset and have the potential to guide key improvements for the next generation of anti-tick vaccines. Team member Dr. Juan Mosqueda published a comparison of diversity within two highly conserved targets, VDAC and subolesin (Perez-Soria et al. 2019. Nova Scientia 11:126-142). He then vaccinated cattle with four conserved peptides from VDAC, and found one peptide that led to a consistent antibody response. The VDAC peptide is a promising candidate that needs to be validated in a larger experiment to tests its ability to control live R. microplus ticks. Anti-tick vaccines will likely become an important tool for APHIS and TAHC tick managers in the near future because of the increase of acaricide resistance in Mexico and Texas.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: GM Klafke, RJ Miller, JP Tidwell, DB Thomas, D Sanchez, TPF Arroyo, and AAP de Leon. High-resolution melt (HRM) analysis for detection of SNPs associated with pyrethroid resistance in the southern cattle fever tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) International Journal for Parasitology-Drugs and Drug Resistance. 2019 Vol. 9 Pages 100-111
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Thomas DB, G Klafke, JD Busch, PU Olafson, RA Miller, J Mosqueda, NE Stone, G Scoles, DM Wagner, A Perez de Leon. Tracking the increase of acaricide resistance in an invasive population of cattle fever ticks (Acari: Iodidae) and implementation of real-time PCR assays to rapidly genotype new resistance mutations. 2020. Annals of the Entomological Society of America 113(4):298-309.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: MME P�rez Soria, DJ Hern�ndez Silva, and J Mosqueda. 2019. Analysis of the allelic variability of BmVDAC and Subolesin, two vaccine candidates against Rhipicephalus microplus in isolates from Mexico. Nova Scientia 11(2):126-142 [In Spanish]
  • Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Mar�a Martina Esperanza P�rez Soria. 2020. Desarrollo y evaluaci�n de una vacuna multiantig�nica y multiepit�pica contra garrapatas Rhipicephalus microplus. Doctoral Dissertation (in Spanish). Universidad Aut�noma de Quer�taro. Facultad de Ciencias Naturales. Dr. Juan Joel Mosqueda Gualito (advisor). [In Spanish]
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Busch JD, J Mosqueda, NE Stone, RE Turner, MPomeroy, SM Hutton, GM Klafke, DB Thomas, G Buckmeier, PU Olafson, GA Scoles, and DM Wagner. Preventing bovine babesiosis: genetic tools for characterizing acaricide resistance and anti-tick vaccines in cattle fever ticks. 99th Annual Conference of Research Workers in Animal Diseases, Chicago Downtown Marriott, Chicago, IL, December 2, 2018. (Talk)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Turner RE, NE Stone, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. 2019. Evaluating genetic variation at an anti-tick vaccine locus to improve eradication of cattle fever ticks. American Society for Microbiology, Arizona and Southern Nevada Branch, Flagstaff, AZ, April 2019. (Talk)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Turner RE, NE Stone, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. 2019. Evaluating genetic variation at an anti-tick vaccine locus to improve eradication of cattle fever ticks. NAU Hooper Undergraduate Research Award Symposium, Flagstaff, AZ, April 2019. (Poster)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Schmidt BA, NE Stone, SM Hutton, J Mosqueda, DB Thomas, GA Scoles, G Buckmeier, PU Olafson, DM Wagner, and JD Busch. An invasive cattle tick in Texas may be a vector of the horse parasite Theileria haneyi. American Society for Microbiology, Arizona and Southern Nevada Branch, Flagstaff, AZ, April 2019. (Poster)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Busch JD, Roberts ML, RE Turner, NE Stone, SM Hutton, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, and DM Wagner. Evaluating genetic variation at anti-tick vaccines to improve cattle fever tick eradication. 2019 Conference of Research Workers in Animal Diseases, 100th Annual Meeting. Chicago Downtown Marriott, Chicago, IL, November 3, 2019. Parasitology Session. (Talk)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Roberts ML, RE Turner, NE Stone, SM Hutton, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. Assessing genetic variation of critical tick genes for anti-tick vaccine development to reduce the spread of cattle fever. Northern Arizona University Undergraduate Research Symposium, Flagstaff, AZ, April 24, 2020. (virtual Poster presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Busch JD, SM Hutton, M Roberts, NE Stone, J Mosqueda, GA Scoles, DB Thomas, GM Klafke, PU Olafson, and DM Wagner. Evaluating genetic variation at anti-tick vaccines to improve cattle fever tick eradication. 2020 Conference of Research Workers in Animal Diseases, 101st Annual Meeting. Chicago, IL (virtual conference due to COVID-19), December 4-8, 2020. Parasitology Session. (Virtual oral presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Roberts ML, RE Turner, NE Stone, SM Hutton, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. Leveraging global genetic diversity to develop more robust cattle fever tick vaccines. Northern Arizona University Undergraduate Research Symposium, Flagstaff, AZ, April 23, 2021. (Poster)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Busch JD, SM Hutton, M Roberts, NE Stone, J Mosqueda, GA Scoles, DB Thomas, GM Klafke, PU Olafson, and DM Wagner. A high level of cryptic cattle fever tick movement occurs in southern Texas via both infested cattle and wildlife. 2021 Conference of Research Workers in Animal Diseases, 102nd Annual Meeting. Chicago, IL, December 3-7, 2021. Parasitology Session. (Virtual oral presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Four members of our scientific team were invited speakers at the 4th Simposio de Garrapatas y Enfermedades que Transmiten (Fourth Symposium on Ticks and Tick-Borne Diseases, held October 19-20, 2021 in Oaxaca City, Oaxaca, Mexico. Online virtual conference in 2021 due to COVID. Busch JD et al. 2021. Molecular genetic tools lead to new insights for managing cattle fever ticks. Ueti M et al. 2021. Targeting Babesia stage-specific proteins as candidates for preventing disease or blocking parasite transmission. Guilherme Klafke GM et al. 2021. Multiple acaracide resistance in Rhipicephalus microplus ticks in Brazil. Mosqueda J et al. 2021. Prote�nas quim�ricas recombinants multiepit�picas: una plataforma para el desarrollo de vacunas contra garrapatas y hemoprotozoarios. [In Spanish]
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Mosqueda J, L�pez DDG, Ortega SR, Camacho NM, P�rez SMM, Casta�eda OJE, Hern�ndez SDJ, and Aguilar TG. Rhipicephalus microplus VDAC is a vaccine candidate that contains conserved B-cell epitopes, which induce antibodies in immunized cattle. BioTicks 2022: The Challenge of Tick Control. 2nd International meeting held in Varadero, Matanzas, Cuba March 27-31, 2022.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: L�pez DDG, P�rez SMM, Ocampo JR, and Mosqueda J. 2022. Evaluaci�n de la immunogenicidad de p�ptidos de tres ant�genos de R. microplus y su efecto sobre par�metros biol�gicos. Proceedings of the 44th National Congress of Buiatrics 2022, hosted by the Mexican Association of Cattle Specialist Veterinarians. Held at the Conference Center for Special Workshops Development (Desarrollo Especial Talleres F.F.C.C.) in Aguascalientes, Mexico on August 4-6, 2022. [In Spanish]


Progress 07/01/20 to 06/30/21

Outputs
Target Audience:Our target audiences were stakeholders in the cattle fever tick eradication program (CFTEP), including landowners, USDA-APHIS inspectors (including tick riders), the TX Animal Health Commission (TAHC), and the wider community of researchers that study Rhipicephalus ticks and Babesia parasites. In Year 3 we worked closely with CFTEP personnel in APHIS and TAHC. We also presented our research ticks at the 2020 Conference of Research Workers in Animal Diseases (CRWAD) held virtually from Chicago, IL, December 4-8, 2020 (Drs. Joe Busch and Massaro Ueti). This project has provided excellent opportunities for scientific training to the students and technical staff at NAU, including Mackenzie Roberts and Shelby Hutton. Shelby was a technician and master's student in our lab who was hired specifically to generate data for this project. They have each worked on the tick project for >2 years and this experience has been a major benefit when they finish their degrees and move on to the next step of their professional training. Changes/Problems:The COVID-19 pandemic continued to slow progress during Y3 and significantly impacted our work at NAU, UAQ, and all USDA-ARS labs. Despite this, we were able to make substantial progress towards Goal 1 in the search for new mechanisms of acaricide resistance. We successfully generated RNAseq data from all 4 resistant colonies of laboratory ticks at USDA, including amitraz, fipronil, coumaphos, and ivermectin. We decided to sequence only 3 larval pools per strain (instead of 6), and so far it appears that we are able to successfully identify major patterns of differential gene expression in resistant versus susceptible ticks. For Goal #2, we have been using AmpSeq to sequence exons from genes that are anti-tick vaccine candidates. One unexpected problem was that our exon-specific primers do not always amplify well in diverse tick samples from Mexico, Brazil, and Colombia. This is likely due to single-nucleotide polymorphisms (SNPs) in the PCR priming sites. While inconvenient, this is consistent with the high level of variation that we are seeing across two tick genes (Bm86 and MP4) that we have sequenced with AmpSeq methodology. What opportunities for training and professional development has the project provided?Our team was involved with several opportunities for professional development during Year 3, including the 2020 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, December 4-8, 2020 (Drs. Joe Busch and Massaro Ueti), and the USDA-ARS Grand Challenge recurring meetings in 2021 led by Drs. Massaro Ueti and Pia Olafson. Both meetings provided excellent opportunities to network with scientists from multiple disciplines and discuss tick management issues. The project has led to valuable professional development for NAU technicians Nate Stone, Shelby Hutton, and Ryelan McDonough who generated all of the RNAseq and AmpSeq data for this study. Nate and Shelby provided professional training for five undergraduate students at NAU who genotyped ticks to search for acaricide resistance mutations and develop our next-generation sequencing methods to investigate vaccine targets. Undergraduate Mackenzie Roberts designed DNA assays for sequencing 13 anti-tick vaccine gene targets and learned valuable skills with generating data, organizing the growing tick database, and conducting safe work in the laboratory. Two technicians from Dr. Mosqueda's research group, Aldo Pavon and Diego Hernandez-Silva, worked on tick colonies and molecular biology for this project at UAQ in Queretaro, MX. How have the results been disseminated to communities of interest? In Year 3, we disseminated information directly to CFTEP personnel in APHIS and TAHC via teleconferences and email updates. Disseminating this knowledge to APHIS will help refine strategies for eradicating ticks on chronically infested premises and increasing surveillance efforts for transported cattle. Presentation at scientific conferences included the 2020 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, December 4-8, 2020 (presentations by Drs. Joe Busch and Massaro Ueti). This meeting is attended by many agricultural scientists and is an important venue interacting with parasitologists involved with the cattle fever tick and babesiosis problem in the Americas and other countries of the world. Publications include: DB Thomas, G Klafke, JD Busch, PU Olafson, RA Miller, J Mosqueda, NE Stone, G Scoles, DM Wagner, A Perez de Leon. Tracking the increase of acaricide resistance in an invasive population of cattle fever ticks (Acari: Iodidae) and implementation of real-time PCR assays to rapidly genotype new resistance mutations. 2020. Annals of the Entomological Society of America 113(4):298-309. What do you plan to do during the next reporting period to accomplish the goals?In Year 4 (a 12-mo no-cost extension period), we will continue making progress on our key goals: • Continue bioinformatic analysis of the RNAseq data from larvae of resistant tick colonies reared at CFTRL in Mission, TX. • Identify gene expression or new SNPs that are associated with resistance to coumaphos, amitraz, fipronil, and ivermectin. • Develop our AmpSeq panels to sequence the exons of 13 gene candidates for anti-tick vaccines. • Write manuscripts and submit findings to high impact scientific journals for publishing.

Impacts
What was accomplished under these goals? OVERALL IMPACTS The highly invasive southern cattle tick (Rhipicephalus microplus) has spread to many regions of the world and transmits parasites that cause cattle fever (bovine babesiosis). This disease and tick vector now occur throughout Mexico and present a constant risk of introduction into southern Texas. The US cattle fever tick eradication control program (CFTEP) currently uses acaricide chemicals to eliminate tick populations that cross the Rio Grande and enter the US on infested stray cattle and wildlife; however, we are in danger of losing this management tool because resistance to all six major classes of tick acaricides has evolved in MX and is moving into the US. The genetic mechanism underlying resistance in cattle fever ticks has only been fully characterized for one chemical class of acaricides, the synthetic pyrethroids. Our research directly addresses this gap in knowledge by comparing differences in gene expression between susceptible and resistant ticks using acaricides from additional chemical classes (coumaphos, amitraz, fipronil, and ivermectin). Once we identify the new mechanisms of resistance for these chemicals, we will be able to design new assays to rapidly screen future tick infestations in Texas for any sign of resistance to each chemical. Our first new assays have been focused on a gene that conveys resistance to synthetic pyrethroids. After screening R. microplus populations from 16 states throughout Mexico we discovered three new mutations for synthetic pyrethroids as well as two previously described mutations. We have been tracking the spread of these mutations from Mexico into Texas since 2017 and observed that over 60% of new tick infestations in Texas carry resistance mutations for synthetic pyrethroids. As such, this project has the potential to inform CFTEP management decisions and extend the useful life of acaricides as an eradication tool. For instance, Texas ranchers should not use synthetic pyrethroids to treat cattle for southern cattle ticks, except in Webb and Zapata Counties where most ticks are susceptible. We have also tested R. microplus populations in Puerto Rico and found that two populations on the western side of the island remain fully susceptible, while a population on the eastern side (near Yabucoa) is highly resistant. Synthetic pyrethroids should only be used in areas where ticks are known to be fully susceptible, or else this chemical will lose its effectiveness in Puerto Rico and Texas within a few more years. The second major impact from our project will be improvements in the development of future anti-tick vaccines for cattle. More than 15 candidate tick proteins have been used experimentally in cattle to raise an antibody response against feeding ticks, but almost nothing is known about genetic variation at the genes encoding these proteins. The one vaccine that is commercially available in the US is based on the Bm86 protein, and previous studies suggest it is unreliable for controlling cattle fever ticks in North America. The problem is due to novel mutations in the Bm86 gene that exist in R. microplus populations in Mexico and the US. By sequencing the Bm86 gene and 14 other vaccine candidates, our study is providing critical baseline information that will improve the next generation of anti-tick vaccines. Members of our scientific team in the US (Dr. Massaro Ueti) and Mexico (Dr. Juan Mosqueda) have been using this new information to design new vaccines based on the most conserved peptides of 5 tick genes. Their current cattle vaccination studies will validate which specific peptides, or combination of peptides, raise a strong antibody response in cattle. After the complete these initial studies, they will proceed to testing tick survival on vaccinated cattle. Anti-tick vaccines will likely become an important tool for tick management because of the increase of acaricide resistance in Mexico and southern Texas. Our new data will be valuable to cattle producers in the Texas eradication zone who are required to use the Bm86 vaccine to reduce tick burdens in cattle herds on the Rio Grande border. Our results will also have impacts for APHIS and TAHC personnel who manage the control of southern cattle ticks in the US. The main accomplishments in Year 3 were: Goal #1. Characterize new genetic mechanisms of resistance for commonly used acaricide chemicals (Y3 90% complete) To advance our search for new acaricide resistance mechanisms, our top priority in Y3 was to generate transcriptome sequences using RNAseq technology for 5 tick colonies maintained at the CFTRL lab in Edinburg, TX. Four of these tick colonies are resistant lineages and the fifth is a fully susceptible control lineage (Deutsch), which was recently used by the USDA to generate a whole genome sequence. Larvae from the appropriate resistant strains were exposed to four chemicals of interest to the CFTEP: amitraz, coumaphos, fipronil, and ivermectin. We now have large transcriptome datasets for each tick colony, and have been performing bioinformatic analyses to identify genes with the greatest level of differential expression. Our first analysis has focused on ticks resistant to amitraz. We are looking at differential expression (DE) in new genes from our project, as well as genes invested in previous publications. Significant new results from Y3 include: We have identified over 50 genes that are differentially expressed (DE) at high level between susceptible and resistant larvae. Five P450 genes are highly DE Several ABC transporters are highly DE Goal #2: Identify genetic variation in tick genes coding for antigens that are high priority candidates as targets for current and future anti-tick vaccines. (Y3 75% complete) In this goal, we analyzed genetic variation at four genes in a large set (n=92) of R. microplus samples from North America, as well as a smaller number from Puerto Rico, Brazil and Colombia. The four genes include Bm86, reprolysin (or MP4), Aquaporin-2 (AQP2), and Vitellogenin Receptor (VitR). Our new sequence data reveal a large number of nucleotide mutations that lead to amino acid changes in the Bm86 and MP4 genes. However, both genes contain short segments that are highly conserved, and we are looking at these conserved regions as potential targets for future vaccine development. Variation was fairly low in VitR and the AQP2 gene is highly conserved across its entire length. These preliminary data show great promise for understanding genetic diversity within anti-tick vaccine genes, which was previously unknown.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Thomas DB, G Klafke, JD Busch, PU Olafson, RA Miller, J Mosqueda, NE Stone, G Scoles, DM Wagner, A Perez de Leon. Tracking the increase of acaricide resistance in an invasive population of cattle fever ticks (Acari: Iodidae) and implementation of real-time PCR assays to rapidly genotype new resistance mutations. 2020. Annals of the Entomological Society of America 113(4):298-309. July 2020 issue.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Busch JD, SM Hutton, M Roberts, NE Stone, J Mosqueda, GA Scoles, DB Thomas, GM Klafke, PU Olafson, and DM Wagner. Evaluating genetic variation at anti-tick vaccines to improve cattle fever tick eradication. 2020 Conference of Research Workers in Animal Diseases, 100th Annual Meeting. Chicago, IL (virtual conference due to COVID-19), December 4-8, 2020. Parasitology Session. (Virtual oral presentation)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2021 Citation: Roberts ML, RE Turner, NE Stone, SM Hutton, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. Leveraging global genetic diversity to develop more robust cattle fever tick vaccines. Northern Arizona University Undergraduate Research Symposium, Flagstaff, AZ, April 23, 2021. (Poster)


Progress 07/01/19 to 06/30/20

Outputs
Target Audience:Our target audiences were stakeholders in the cattle fever tick eradication program (CFTEP), including landowners, USDA-APHIS inspectors (including tick riders), the TX Animal Health Commission (TAHC), and the wider community of researchers that study Rhipicephalus ticks and Babesia parasites. In Year 2 we have been working closely with CFTEP personnel in APHIS and TAHC. At the request of the CFTEP National Program Leader, Dr. Denise Bonilla, we presented updates from our genetic analyses to over 100 tick management personnel in Texas at two teleconferences (April 3rd and June 3rd, 2020). We also reached out to researchers of ticks and tick-borne diseases at scientific conferences. This includes the 2019 Congress for the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in Madison, WI, July 7-11, 2019 (Drs. Joe Busch, Donald Thomas, and Guilherme Klafke) and the 100th annual Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, November 2-5, 2019 (Drs. Joe Busch and Massaro Ueti). Both meetings provided excellent opportunities to network with our international target audience and led to insightful discussions with tick management personnel from other countries. WAAVP in particular is an important international audience for management personnel and scientists around the world. This project has provided excellent opportunities for scientific training to the students and technical staff at NAU, including Mackenzie Roberts, Shelby Hutton, and Ashley Jones. All of our past students at NAU were hired specifically to generate data for this specific project. They have each worked on the tick project for 1-2 years and this experience will be a major benefit when they finish their undergraduate degrees and move on to the next step of their professional training. Changes/Problems:Two significant challenges have delayed our progress in Y2. First, NAU was unable to transferfunds to UAQ, Mexico, for our subcontract with Juan Mosquedadue to issues with US Federal SAM registration. This issue continued for 16 months, until Y2 was halfway finished. After many frustrating attempts, Dr. Mosqueda was able to successfully register for SAM and the funds have been transferred to UAQ. The second major challenge was the COVID-19 pandemic. This affected work at NAU, UAQ, and all USDA-ARS labs. At NAU, our technical staff were diverted to other research projects focused on COVID, and we have not been able to make progress in our search for new mechanisms of acaricide resistance using RNAseq analysis. Fortunately, we still have funding available for this goal and will request a 12-month no-cost extension to complete the unfinished work. What opportunities for training and professional development has the project provided?Our team was involved with several opportunities for professional development during Year 1, including the 2019 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, November 2-5, 2019 (Drs. Joe Busch and Massaro Ueti), and the 2019 Congress for the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in Madison, WI, July 7-11, 2019 (Drs. Joe Busch, Don Thomas, and Gui Klafke). Both meetings provided excellent opportunities to network with scientists from other countries and discuss tick management issues. The project has led to valuable professional development for NAU technicians Nate Stone and Shelby Hutton who conduct all of the genotyping for this study. Nate and Shelby provided professional training for five undergraduate students at NAU who genotyped ticks to search for acaricide resistance mutations and develop our next-generation sequencing methods to investigate vaccine targets. Our students learned valuable skills in generating data, organizing the large tick database, and conducting safe work in the laboratory. Undergraduate Mackenzie Roberts designed DNA assays for sequencing 13 anti-tick vaccine gene targets. Two technicians in Dr. Mosqueda's research group worked on tick colonies at UAQ and molecular biology for this project, Aldo Pavon and Diego Hernandez-Silva. In addition, two of Dr. Mosqueda's graduate students at UAQ finished their degrees as part of this project: Martina Perez Soria (PhD degree) and Rodrigo Morales (MS degree). How have the results been disseminated to communities of interest?In Year 2, we disseminated information directly to CFTEP personnel in APHIS and TAHC via teleconferences and email updates. Disseminating this knowledge directly to APHIS will help refine strategies for eradicating ticks on chronically infested premises and increasing surveillance efforts for transported cattle. Team members also presented at multiple scientific meetings. At the request of the CFTEP National Program Leader, Dr. Denise Bonilla, we presented updates from our genetic analyses to about 100 tick management personnel in Texas during two teleconferences (April 3rd and June 3rd, 2020) that included question and answer sessions. Presentations at scientific conferences include: • 2019 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, November 1-4, 2019 (presentations by Drs. Joe Busch and Massaro Ueti) • 2019 Congress for the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in Madison, WI, July 7-11, 2019 (presentations by Drs. Don Thomas and Gui Klafke). Both meetings were important venues for interacting with parasitologists involved with the cattle fever tick and babesiosis problem in the Americas and other countries of the world. Publications include: DB Thomas, G Klafke, JD Busch, PU Olafson, RA Miller, J Mosqueda, NE Stone, G Scoles, DM Wagner, A Perez de Leon. Tracking the increase of acaricide resistance in an invasive population of cattle fever ticks (Acari: Iodidae) and implementation of real-time PCR assays to rapidly genotype new resistance mutations. 2020. Annals of the Entomological Society of America 113(4):298-309. What do you plan to do during the next reporting period to accomplish the goals?In Year 3, we will continue making progress on our key goals: • Perform transcriptome analysis on the RNA samples that Dr. Gui Klafke provided from larvae of resistant tick colonies reared at CFTRL in Mission, TX. • Identify gene expression or new SNPs that are associated with resistance to coumaphos, amitraz, fipronil, and ivermectin. • Develop our AmpSeq panels to sequence the exons of all 15 gene candidates for anti-tick vaccines. • Write manuscripts and submit findings to high impact scientific journals for publishing.

Impacts
What was accomplished under these goals? OVERALL IMPACTS Preventing the re-introduction of cattle fever (bovine babesiosis) from Mexico can only be achieved by eradicating cattle fever ticks in southern Texas. The cattle fever tick eradication control program (CFTEP) currently depends on the use of acaricide chemicals to eliminate tick populations, and we are in danger of losing this management tool because resistance to the six major classes of acaricides has arisen in MX and is moving into the US. Genetic mechanisms of resistance in cattle fever ticks have been fully characterized for just one chemical class of acaricides, the synthetic pyrethroids. Our research directly addresses this gap in knowledge by using gene expression analysis to identify new mechanisms of resistance to four chemicals that are important for tick control (coumaphos, amitraz, fipronil, and ivermectin). Once the mechanisms have been identified, new assays can be developed to rapidly screen future tick infestations in Texas for mutations that convey resistance to these chemicals. This project has the potential to inform CFTEP management decisions and extend the useful life of acaricides as an eradication tool. As an example, in this NIFA-funded projectwe found resistance mutations for synthetic pyrethroids in cattle fever tick populationsthroughout Mexico. These mutations have spread into Texas during the past 10 years and since 2017, over 50% of the new tick infestations in Texas are resistant to synthetic pyrethroids.Therefore, Texas ranchers should not use this chemical class to control ticks if their cattle become infested. In contrast, the tick populations we have tested in Puerto Rico remain fully susceptible to most acaricides, and cattle producers can continue the use of synthetic pyrethroids. Our project also has the potential to impact the development of future anti-tick vaccines. More than 15 candidate proteins from ticks have been used experimentally in cattle to raise an antibody response against R. microplus ticks, but almost nothing is known about genetic variation at the genes encoding these proteins. Only one vaccine is commercially available in the US; it is based on the Bm86 protein and previous studies suggest its ability to control ticks in North America varies widely, possibly because of novel mutations in the Bm86 gene that occur in R. microplus populations from Mexico and the US. By sequencing the Bm86 gene and 14 other vaccine candidates, our study will provide baseline information needed to improve the next generation of anti-tick vaccines. These vaccines will become an important tool for tick management if resistance mutations from Mexico begin to spread rapidly in southern Texas. Our new datawill be valuable to eradication personnel (APHIS and TAHC) who are currently using the Bm86 vaccine to supplement acaricides as a way to control ticks. Our project will also have impacts for cattle producers in the US, especially small operations in southern Texas where ticks repeatedly infest cattle. Goal #1. Characterize new genetic mechanisms of resistance for commonly used acaricide chemicals (Y2 60% complete) To advance our search for new acaricide resistance mechanisms, one of our top priorities in Y1 was to generate RNA samples from 6tick colonies maintained at the CFTRL lab in Mission, TX. Five of these tick colonies are resistant lineages and the sixth is a fully susceptible control lineage. Resistant larvae from these colonies were exposed to four chemicals of interest to the CFTEP: amitraz, coumaphos, fipronil, and ivermectin. We used a fifth chemical, permethrin, because its resistance mechanism is already fully described in ticks and other arthropods. After exposure to acaricides, we extracted RNA from pools of larvae that will be used for RNAseq transcriptome sequencing in Y3. We also performed biochemical assays on these ticks to investigate which metabolic pathways might possibly contribute to resistance. In a related effort, we screened >1,500 ticks from field collections in Mexico and the US to search for new mutations in the gene responsible for resistance to synthetic pyrethroids, the para-sodium ion channel. We found two new SNPs in this gene, one at nucleotide position 215 and another at 2136. The G215T SNP has been previously described in Australia but was not known to occur in North America until our study; it appears to be rare and we found it in only two Mexican states (Campeche and Queretaro). The C2136A SNP has not been described yet in R. microplus, but is located in domain II of the sodium ion channel within an important transmembrane region where numerous resistance mutations have already been described in many insect pests. Thus, there is a reasonable chance that it could play a role in resistance and this can easily be tested with larval packet testing. SNP C2136A is uncommon in Mexico but spread throughout the country. We have not yet found these two SNPs in Texas, which is good news for US cattle producers. Documenting new SNPs will complement our transcriptome experiments and help us identify new mechanisms of acaricide resistance in R. microplus. Goal #2: Identify genetic variation in tick genes coding for antigens that are high priority candidates as targets for current and future anti-tick vaccines. (Y2 25% complete) In this goal, we analyzed genetic variation in several genes that are candidates for anti-tick vaccines: Bm86, reprolysin (or MP4), and 3 serpin genes. We have been optimizing our next-generation amplicon sequencing on the MiSeq Illumina platform for each gene target. We have sequence data for a large set (n=92) of R. microplus samples from North America, as well as a smaller number from Puerto Rico and South America (Brazil and Colombia). These new data reveal a large number of mutations in the Bm86 and MP4 genes. Each gene contains a few short segments that are highly conserved, and we are looking at these conserved regions as potential targets for future vaccine development. Our preliminary data show great promise for understanding novel genetic diversity within anti-tick vaccine genes.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: DB Thomas, G Klafke, JD Busch, PU Olafson, RA Miller, J Mosqueda, NE Stone, G Scoles, DM Wagner, A Perez de Leon. Tracking the increase of acaricide resistance in an invasive population of cattle fever ticks (Acari: Iodidae) and implementation of real-time PCR assays to rapidly genotype new resistance mutations. 2020. Annals of the Entomological Society of America 113(4):298-309
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: J.D. Busch, R.E. Turner, N.E. Stone, M. Roberts, G.M. Klafke, P.U. Olafson, G.A. Scoles, J. Mosqueda, D.B. Thomas, D.M. Wagner. Evaluating genetic variation at anti-tick vaccines to improve cattle fever tick eradication. 2019 Conference of Research Workers in Animal Diseases, 100th Annual Meeting. Chicago Downtown Marriott, Chicago, IL, November 3, 2019. Parasitology Session. (Talk)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: M.L. Roberts, R.E. Turner, N.E. Stone, S.M. Hutton, J. Mosqueda, D.B. Thomas, P.U. Olafson, G.A. Scoles, D.M. Wagner, and J.D. Busch. Assessing genetic variation of critical tick genes for anti-tick vaccine development to reduce the spread of cattle fever. Northern Arizona University Undergraduate Research Symposium, Flagstaff, AZ, April 24, 2020. (Poster)


Progress 07/01/18 to 06/30/19

Outputs
Target Audience:Our target audiences were stakeholders in the cattle fever tick eradication program (CFTEP), including landowners, USDA-APHIS inspectors (including tick riders), the TX Animal Health Commission (TAHC), and the wider community of researchers that study Rhipicephalus ticks and Babesia parasites. In Year 1, we have reached out to researchers of ticks and tick-borne diseases at scientific conferences. This includes the 2018 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, December 1-4, 2018 (Dr. Joe Busch), and the 2019 Congress for the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in Madison, WI, July 7-11, 2019 (Drs. Joe Busch, Donald Thomas, and Guilherme Klafke). Both meetings provided excellent opportunities to network with our international target audience and led to insightful discussions with tick management personnel from other countries. WAAVP in particular is an important international audience for management personnel and scientists around the world. This project has provided excellent opportunities for scientific training to the students and technical staff at NAU, including Rebekah Turner, Shelby Hutton, Bryce Schmidt, Mackenzie Roberts, and Ashley Jones. All of our students at NAU were hired specifically to generate data for this specific project. They have each worked on the tick project for 1-2 years and this experience will be a major benefit when they finish their undergraduate degrees and move on to the next step of their professional training. Changes/Problems:A challenge in Year 1 has been to obtain R. microplus DNA samples from Africa and Asia. To address this, we are reaching out to our network of international collaborators to obtain the desired tick samples. We have verbal commitments from scientists in several countries who will ship DNA to us in Year 2. Fortunately, we have successfully obtained ticks from Brazil through Dr. Guilherme Klafke's collaborators, and ticks from Pakistan through Dr. Shahid Karim, a collaborator of Dr. Glen Scoles. In Year 1 we have also experienced a delay in preparing RNA samples from certain lab colonies of R. microplus reared at the CFTRL in Mission, TX. We decided it was important to subject tick colonies to acaricides for several generations to insure we were working with highly resistant genotypes. Dr. Kalfke has completed the selection process and sent 125 RNA samples to NAU at the end of Year 1. NAU is now moving forward with the RNA transcriptome study as planned. What opportunities for training and professional development has the project provided?Our team was involved with several opportunities for professional development during Year 1, including the 2018 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, December 1-4, 2018 (Drs. Joe Busch and Massaro Ueti), and the 2019 Congress for the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in Madison, WI, July 7-11, 2019 (Drs. Joe Busch, Don Thomas, and Gui Klafke). Both meetings provided excellent opportunities to network with scientists from other countries and discuss tick management issues. The project has led to valuable professional development for NAU technicians Nate Stone and Shelby Hutton who conduct all of the genotyping for this study. Nate and Shelby provided professional training for three undergraduate students at NAU who are genotyping ticks to search for acaricide resistance mutations and develop our next-generation sequencing methods to investigate vaccine targets. Our students learned valuable skills in generating data, organizing our tick database, and working safely in the laboratory. Undergraduate Rebekah Turner designed DNA assays for sequencing the anti-tick vaccine gene target known as Bm86. She earned 3 awards during this project, including a Hooper Undergraduate Research Award (HURA) from NAU. Her HURA project was eventually selected for the prestigious Posters on the Hill conference held in Washington, DC in April 2019. At this conference she met Peter Johnson, the USDA-NIFA National Program Leader, who gave her advice on the project and discussed possible funding sources for her future graduate degree. The work started by Ms. Turner is being continued by a new undergraduate, Mackenzie Roberts. The third student on this project was Bryce Schmidt, who screened ticks for resistance mutations using qPCR assays. How have the results been disseminated to communities of interest?In Year 1, our team members presented at two scientific meetings, including: 2018 Conference of Research Workers in Animal Diseases (CRWAD) held in Chicago, IL, December 1-4, 2018 (presentations by Drs. Joe Busch and Massaro Ueti) 2019 Congress for the World Association for the Advancement of Veterinary Parasitology (WAAVP) held in Madison, WI, July 7-11, 2019 (presentations by Drs. Don Thomas and Gui Klafke). Both meetings were important venues for interacting with parasitologists involved with the cattle fever tick and babesiosis problem in the Americas and other countries of the world. We have also disseminated our results with personnel from USDA-ARS and APHIS. Disseminating this knowledge directly to APHIS will help refine strategies for eradicating ticks on chronically infested premises and increasing surveillance efforts for transported cattle. What do you plan to do during the next reporting period to accomplish the goals?In Year 2, we will continue making progress on our key goals: Perform transcriptome analysis on the RNA samples that Dr. Gui Klafke provided from larvae of resistant tick colonies reared at CFTRL in Mission, TX. Screen Texas tick collections for new SNPs in the para-sodium ion channel that are associated with resistance Develop our AmpSeq panels to sequence the exons of 10 genes that are candidates for anti-tick vaccines

Impacts
What was accomplished under these goals? The main accomplishments in Year 1 were the following: Goal #1. Acaricide resistance (Y1 30% complete) To advance our search for new acaricide resistance mechanisms, one of our top priorities in Y1 was to generate RNA samples from 6 tick colonies maintained at the CFTRL lab in Mission, TX. Five of these tick colonies are resistant lineages and the sixth is a fully susceptible control lineage. Resistant larvae were exposed to the four chemicals used in this study: amitraz, coumaphos, ivermectin, and permethrin. After exposure to acaricides, we extracted RNA from pools of larvae that will be used for RNAseq transcriptome sequencing in Y2. We also performed biochemical assays on these ticks to investigate which metabolic pathways might possibly contribute to resistance. In a related effort, we also screened >1,500 ticks from field collections in Mexico to search for new mutations in the gene responsible for resistance to synthetic pyrethroids, the para-sodium ion channel. We found two new SNPs in this gene, one at nucleotide position 215 and another at 2136. The G215T SNP has been previously described in Australia but was not known to occur in North America until our study; appears to be rare and we found it in only two Mexican states (Campeche and Queretaro). The C2136A SNP has not been described yet in R. microplus, but is located in domain II of the sodium ion channel within an important transmembrane region where numerous resistance mutations have already been described in many insect pests. Thus, there is a reasonable chance that it could play a role in resistance and this can easily be tested with larval packet testing. SNP C2136A is uncommon in Mexico occurs throughout the country. It is unknown whether these two SNPs have reached Texas because we still need to screen Texas samples. Documenting new SNPs will complement our transcriptome experiments and help us identify new mechanisms of acaricide resistance in R. microplus. Goal #2: Anti-tick vaccines (Y1 10% complete) In this goal, we analyzed genetic variation in several genes that are candidates for anti-tick vaccines: Bm86, reprolysin (or MP4), and 3 serpin genes. We are in the process of optimizing our ampseq PCR and next-generation amplicon sequencing on an Illumina platform (MiSeq) for these gene targets. First, we design and test primers against a small set (n=7) of diverse R. microplus and R. annulatus. Even with this small number of ticks samples, we have discovered a large number of mutations in the Bm86 and reprolysin genes. These mutations are spread across the full length of both genes. However, each gene does contain a few short segments that are highly conserved, even between R. microplus and R. annulatus. It is possible that these conserved regions could become a target for future vaccine development. Our preliminary data show great promise for documenting diversity within anti-tick vaccine genes. OVERALL IMPACTS Our new data on acaricide resistance mutations have already increased the understanding of cattle fever tick populations in the US and Mexico. Our findings demonstrate that new resistance mutations are continually arising in tick populations from North America. We have found new mutations that appear to be associated with resistance to synthetic pyrethroids, and also verified the existence of potential SNPs for amitraz and fipronil resistance in Mexico and Texas that were identified by other researchers. Resistance is a major problem in Mexico that is spilling into Texas, and unfortunately the majority of new infestations in Texas since 2017 are resistant to synthetic pyrethroids. Texas ranchers and APHIS tick eradication personnel need to choose appropriate pesticides to eradicate ticks; in most cases synthetic pyrethroids should not be used in Texas. If ticks are treated with an ineffective chemical, then a resistant population will spread on cattle and even infest wildlife hosts like white-tailed deer, which are almost impossible to treat. In contrast, the tick populations we have tested in Puerto Rico remain fully susceptible to most acaricides. This information is valuable to eradication personnel (APHIS and TAHC) who make decisions about preventing bovine babesiosis through tick eradication. Our findings also have impacts for cattle producers in the US, especially small operations in southern Texas where ticks are established. We are still at an early phase of our research and will continue to learn more about cattle fever tick populations in North America by RNA transcriptome sequencing and investigating variation at anti-tick vaccines.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Donald B. Thomas, Guilherme Klafke, Joseph D. Busch, Pia U. Olafson, Robert A. Miller, Juan Mosqueda, Nathan L. Stone, Glen Scoles, David M. Wagner, and Adalberto Perez-de-Leon. Tracking the increase of acaricide resistance in an invasive population of cattle fever ticks, Rhipicephalus (Boophilus) microplus Canestrini (Acari: Ixodidae) and implementation of real-time PCR assays to rapidly genotype resistance mutations. 2019. Annals of the Entomological Society of America (under review)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: J. D. Busch, J. Mosqueda, N. E. Stone, R. E. Turner, M. Pomeroy, S. M. Hutton, D. Thomas, G. Buckmeier, P. U. Olafson, G. A. Scoles, and D. M. Wagner. Preventing bovine babesiosis: genetic tools for characterizing acaricide resistance and anti-tick vaccines in cattle fever ticks. 99th Annual Conference of Research Workers in Animal Diseases, Chicago Downtown Marriott, Chicago, IL, December 2, 2018. (Talk)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bryce Anthony Schmidt, NE Stone, RE Turner, SM Hutton, J Mosqueda, DB Thomas, G Buckmeier, GA Scoles, PU Olafson, DM Wagner, and JD Busch. An invasive cattle tick in Texas may be a vector of the horse parasite Theileria haneyi. American Society for Microbiology, Arizona and Southern Nevada Branch, Flagstaff, AZ, April 2019. (Poster)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Bryce Anthony Schmidt, NE Stone, RE Turner, SM Hutton, J Mosqueda, DB Thomas, G Buckmeier, GA Scoles, PU Olafson, DM Wagner, and JD Busch. An invasive cattle tick in Texas may be a vector of the horse parasite Theileria haneyi. NAU 11th Annual Undergraduate Symposium, Flagstaff, Arizona, April 2019. (Poster)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Rebekah E. Turner, NE Stone, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. 2019. Evaluating genetic variation at an anti-tick vaccine locus to improve eradication of cattle fever ticks. American Society for Microbiology, Arizona and Southern Nevada Branch, Flagstaff, AZ, April 2019. (Talk)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Rebekah E. Turner, NE Stone, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. 2019. Evaluating genetic variation at an anti-tick vaccine locus to improve eradication of cattle fever ticks. NAU Hooper Undergraduate Research Award Symposium, Flagstaff, AZ, April 2019. (Poster)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Rebekah E. Turner, NE Stone, J Mosqueda, DB Thomas, PU Olafson, GA Scoles, DM Wagner, and JD Busch. 2019. Evaluating genetic variation at an anti-tick vaccine locus to improve eradication of cattle fever ticks. Posters on the Hill undergraduate symposium, Washington, DC, April 30, 2019. (Poster)