Location: Tick and Biting Fly Research
2007 Annual Report
Identification of bovine host genes that associate with the tick-resistant phenotype: It is essential to identify and define host genes that are involved in natural tick innate and acquired resistance. DRB3 is a class IIa gene of the bovine leukocyte antigen complex that is being studied. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, analysis of a region of the DRB3 gene from the phenotyped calves in our genomic DNA database herd revealed a significant association between the DRB3*4401 allele and the tick-resistant phenotype. Identification of resistance associated genes will allow for selective markers and enhanced vaccine efficacy. (NP-104; Component 3, Biology and Physiology; Goal 3.1.4)
Analysis of host-pathogen interaction between Babesia bovis and R. microplus: In order to better understand the mechanisms of pathogen transmission it is necessary to compare and contrast gene expression in ovarial and gut tissues of B. bovis infected and uninfected R. microplus. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, the ovarial and gut proteomes of R. microplus adult females infected with R. bovis have been analyzed by 2-D PAGE and specific proteins that are differentially expressed in response to infection have been identified by mass spectrometry and bioinformatic analysis. Differential gene expression in response to R. bovis infection in these tissues has also been analyzed by microarray analysis and subtraction library screening, allowing examination of this critical interaction at the gene transcript and protein level. This information allows for the study of vector competence regarding the transmission of the pathogen, B. bovis, by the tick, R. microplus, to cattle. (NP-104; Component 3, Biology and Physiology; Goal 3.1.1)
Variation within Glucose 6-phosphate dehydrogenase (G6PDH), a tick enzyme in R. microplus: Developing molecular epidemiological techniques to determine genetic relatedness of outbreak tick strains is necessary to provide better support of the APHIS-VS, Cattle Fever Tick Eradication Program. At the Knipling-Bushland U.S. Livestock Insects Research Laboratory, Kerrville, TX, four different transcripts for this enzyme have been identified. In addition, on non-denaturing polyacrylamide gels, more than one protein band with G6PDH activity was observed, suggesting the presence of multiple forms of this enzyme expressed by the tick. We postulate that gender and life-stage differential expression of these transcripts of G6PDH may play a role in tolerance of oxidative stress that is induced upon feeding, and that the transcript abundance in fed females is a function of bloodmeal volume and the time adult females spend on the host relative to adult males. Both male and female ticks require an adequate blood meal to insure successful reproduction. Physiological events that protect ticks while feeding represent targets for control intervention. Identification of those targets is essential to the development of novel control technologies. Molecular tools that can provide information as to the genetic relationships between ticks collected from outbreak sites provide epidemiologists with valuable information for the management and understanding of outbreaks. (NP-104; Component 3, Biology and Physiology; Goal 3.1.1, and NP-104 Component 2, Detection and Surviellance Technology; Goal 2.1.1)
Development of an internal standard for quantitative-PCR (Q-PCR) and quantitative reverse transcriptase-PCR (QRT-PCR) assays in R. microplus: Quantitation of mRNA in different tissues or under different conditions requires an internal invariant reference to use as a normalization standard to allow comparison of expression of the test mRNA. "Housekeeping" genes are generally used for this purpose, but previous studies in other organisms has shown that multiple reference genes are needed, as none have been found to be truly invariant. To date, the only published report of quantitative PCR in R. microplus utilized beta-actin as an internal reference for normalization between samples. Our studies have indicated that there are multiple transcripts or isoforms of beta-actin in R. microplus, making it a poor choice as an internal reference standard. We have developed 18S DNA/RNA as an internal reference standard, chosen because it is a major component of ribosomes, expressed at high levels in most tissues, and is generally known to be under tight transcriptional control. This represents a versatile and useful internal standard for relative quantization within essentially any tissue for genes that are highly expressed, or those expressed at relatively low levels, providing functional genomic researchers with a valuable tool. (NP-104; Component 3, Biology and Physiology; Goal 3.1.1)
Olafson, P.U., Pruett Jr, J.H., Steelman, C.D. 2007. Association of the bovine leukocyte antigen major histocompatibility complex class II DRB3*4401 allele with host resistance to the Lone Star Tick, Amblyomma americanum. Veterinary Parasitology. 145(1-2):190-195.
Guerrero, F.D., Bendele, K.G., Chen, A.C., Li, A.Y., Miller, R.J., Pleasance, E., Varhol, R., Rousseau, M.-E., Nene, V.M. 2007. Serial analysis of gene expression in the southern cattle tick following acaricide treament of larvae from organophosphate resistant and susceptible strains. Insect Molecular Biology. 16(1):49-60.
Guerrero, F., Bendele, K.G., Davey, R.B., George, J.E. 2007. Detection of Babesia bigemina infection in strains of Rhipicephalus (Boophilus) microplus collected from outbreaks in South Texas. Veterinary Parasitology. 145:156-163.