Performing Department
Entomology
Non Technical Summary
Ticks are an ever-present threat to the health of Texas' cattle and other livestock. Ticks are important and competent vectors for a large range of pathogens that cause disease in animals and humans. Additionally, as our science advances, we are better able to describe the impacts that ticks have in causing direct damage from their feeding or indirect damage by the capacity to vector disease-causing pathogens. The research in this project will generate valuable insight on the specific mechanisms as to how a tick can survive an otherwise fatal pesticide exposure, improve the detection of larval and nymphal ticks attached to imported cattle from Mexico (>1M/yr), and provide a deeper understanding of introduced and native tick species' roles in transmitting pathogens important to livestock and human health. From the expected results generated from the research in this project, we can better protect the health of and improve economic return for the cattle and other livestock industries in Texas.
Animal Health Component
100%
Research Effort Categories
Basic
25%
Applied
75%
Developmental
(N/A)
Goals / Objectives
Evaluation of population genetics and pesticide resistance in livestock-associated ticks.Improvement of cattle fever tick monitoring and prevention to enhance protection of the US cattle industry.Determination of the incidence, prevalence, and distribution of existing and introduced ticks and tick-borne pathogens that threaten Texas livestock and horses.
Project Methods
Evaluation of population genetics and pesticide resistance in livestock-associated ticks.Rhipicephalus microplus strains maintained at the USDA-ARS Cattle Fever Tick Research Laboratory that are known to exhibit cross-resistance to acaricides (pyrethroids, amitraz, organophosphates), as well as ivermectin, will be utilized. These strains will be subjected to the larval immersion test to determine the level of resistance to ivermectin (Klafke et al. 2006) and to facilitate phenotypic separation of ticks expressing this resistance (survivors of higher ivermectin concentrations are more likely to express the resistance alleles).RNA will be extracted from individual adult female ticks (susceptible and resistant) followed by cDNA synthesis using standard protocols. The segment containing the putative sequences of the GluCl-channel gene of R. microplus will be amplified using primers designed by Gassel et al. (2014) and the PCR products will then be subjected to Sanger sequencing for characterization of the GluCl-channel gene of R. microplus. The nucleic acid sequences from susceptible and resistant ticks will be translated and aligned to GluCl-channel protein sequences from Tetranycus urticae (Acari: Tetranychidae) to look for the single nucleotide polymorphisms associated with ivermectin resistance previously identified in this mite (Kwon et al. 2010).An HRM assay will be developed to genotype the different ivermectin resistance related polymorphisms in the GluCl-channel gene of R. microplus. The assay will use genomic DNA from individual adult females from susceptible and resistant strains and primers designed based on the partial sequence of the R. microplus glutamate-gated chloride channel gene. HRM results will be confirmed by Sanger sequencing.An RNA-seq comparative transcriptome approach will be used to compare the expression of metabolic detoxification enzymes (CytP450s, ABC transporters) in ivermectin-susceptible and -resistant strains of R. microplus.Improvement of cattle fever tick monitoring and prevention to enhance protection of the US cattle industry.Tick predilection site determination:Six heifers or steers will be artificially infested with 250 mg of R. microplus larvae (recommendation of CFTL personnel) and held in the barn at the USDA-ARS Cattle Fever Tick Research Laboratory throughout the study. At 4-5 days post-infestation, animals will be inspected by a CFTRL caretaker experienced in tick detection and the number and location of larvae found on each animal will be recorded. Three animals will be euthanized on the same day, and their hides will be removed from the carcass manually and cut into 18 sections to facilitate handling and specification of tick locations (Watson et al. 1997). The sections will be individually packed in plastic bags and frozen to kill all ticks and will remain frozen until further examination.To search for attached nymphs and remaining larvae, on days 9-10 post-larval infestation, the remaining three animals will be inspected by the trained inspector again, and the number and location of larvae and nymphs mapped and recorded. The three animals will be euthanized on the same day, their hides recovered, sectioned, and stored as described above.To gather detailed information on tick density, survival (to inspection date) and distribution, the sections of frozen hide will be visually inspected, and the number of larvae and nymphs found in each section will be recorded as well as hair length. Larger sections of hide will likely be sub-sectioned to increase accuracy of tick locations. Following the visual search, the hides will be processed using a modified KOH dissolution technique. The hair of each body section will be lightly moistened with water and the sample sealed in a plastic bag and held at 20°C until the hair slips from the skin. Collected hair and ticks will be dissolved at room temperature with 10% KOH solution for 24-48 h and the ticks will be recovered by filtration.The larvae and nymphs recovered from the hides will be mapped to the hide sections allowing for determination of density and distribution. An overall assessment will be made of percent survival by comparing infestation number to tick recovery number (for each life stage). Using this approach, the accuracy of tactile inspection as performed by a trained inspector will be compared to the number of larvae and nymphs recovered from the hides. Such information will be highly informative for future tick inspections and improve the security of the Texas cattle industry.Determination of the incidence, prevalence, and distribution of existing and introduced ticks and tick-borne pathogens that threaten Texas livestock and horses.This study will include a cooperative effort with involvement by Texas A&M AgriLife Extension entomologists and the Texas Animal Health Commission. We will collect R. sanguineus from cattle at auction markets throughout Texas and ask our collaborators to submit R. sanguineus collected from cattle during passive and active surveillance related to the Cattle Fever Eradication Program. There are 129 livestock markets in Texas, and we plan to visit a great proportion of them through our partners. We anticipate collecting and/or receiving approximately 500 ticks throughout the study. Tick morphological identification will be performed using standard keys (Walker et al. 2000).To determine the prevalence of A. marginale and R. rickettsii in brown dog ticks collected from cattle, nucleic acid will be purified from individual, non-fed nymphs and adult ticks using the MagMAX™ CORE Nucleic Acid Purification Kit and the KingFisher Flex Purification System. The pathogens (A. marginale and R. rickettsii) will be identified by amplification of the following genes: Ankyrin-repeat protein (AnkA), outer membrane protein 8 (omp8), and major surface protein 1B-2 (msp1B-2) for A. marginale (Hairgrove et al., 2015); citrate synthase (gltA), outer membrane protein A (ompA), and outer membrane protein B (ompB) for R. rickettsii (Labruna et al., 2014). To confirm pathogen identification, the qPCR products will be purified and both strands will be directly sequenced by Sanger sequencing by a third party provider. As appropriate, a comparison to the USDA-approved cELISA serologic test for A. marginale detection may be conducted.To identify the lineages and distribution of brown dog tick collected from cattle in Texas, nucleic acid will be purified from individual, non-fed nymphs and adult ticks as described previously for pathogens. The tick lineages (temperate or tropical) will be determined by amplification of the partial mitochondrial 16S ribosomal DNA (16S rDNA) (Mangold et al., 1998) and 12S ribosomal DNA (12S rDNA) (Beati & Keirans, 2001) by qPCR. To confirm lineage identification, the qPCR products will be purified and both strands will be directly sequenced using Sanger sequencing by a third party provider.