Expression of bovine genes associated with local and systemic immune response to infestation with the Lone Star tick, Amblyomma americanum.

The Lone Star tick, Amblyomma americanum Linnaeus 1758 (Acari; Ixodidae), causes considerable production losses to the southern U.S. cattle industry due to reduced weight, infertility, secondary infections at bite wound sites, damaged hides, and potentially death, as these ticks tend to infest livestock in large numbers. Increasing environmental concerns, along with the potential for chemical residue in food products, have led to more emphasis on alternative tick control strategies, such as selective breeding practices and anti-tick vaccines. To enable progress toward these goals, a better understanding of bovine host immune mechanisms elicited by ticks is needed. In this study, 7 calves were phenotyped as susceptible, moderately resistant, or highly resistant to adult A. americanum ticks. Tick bite-site biopsies and blood leukocytes were collected at multiple time points throughout 3 successive tick infestations. Gene expression at tick bite-site biopsies was assessed by microarray analysis over 3 time points for each phenotype group. Quantitative reverse transcriptase-PCR expression analysis evaluated 11 candidate genes in tick bite-site biopsies, and 6 in blood leukocytes. Regression curve estimates calculated from the expression values generated by qRT-PCR in tick bite-sites identified correlations between several candidate genes. Increased expression of IGHG1, IL6, IL1α, and IL1RN in bovine tick bite-site biopsies suggests that Th2 differentiation may be important for the local bovine response to A. americanum ticks. Strong correlations in expression for IL1α and IL1ß, for IL1α and IL1RN, and for IL1α and TLR4 were found in biopsies from the tick-resistant phenotypes. The up-regulation of IL12 and IL23 in blood leukocytes from Lone Star tick-infested calves of all phenotypes suggests a possible systemic recruitment of memory T cells. This study provides novel insight concerning the bovine immune response to Lone Star ticks and a basis for future investigations to characterize the importance of these factors for tick-resistance in cattle.

Evaluation of methods for the isolation of high quality RNA from bovine and cervine hide biopsies.

Molecular investigations of the ruminant response to ectoparasites at the parasite-host interface are critically dependent upon the quality of RNA. The complexity of ruminant skin decreases the capacity to obtain high quality RNA from biopsy samples, which directly affects the reliability of data produced by gene expression experiments. Two methods for isolating total RNA from skin were compared and the use of 4M guanidinium isothiocyanate (GITC) during frozen storage of the specimens was evaluated. In addition, the best procedure for RNA isolation from bovine skin punch biopsies was also tested on white-tailed deer skin biopsies. Skin biopsy punches were collected and frozen prior to pulverization for RNA isolation. Total RNA quantity and integrity were determined by spectrophotometry and capillary electrophoresis technology, respectively. Significantly increased total RNA yield (P < 0.05) and higher integrity (P < 0.05) were obtained with a TRI Reagent® isolation method. Freezing and subsequent storage of bovine skin punch biopsies in 4 M GITC did not affect the amount or integrity of total RNA recovered by either RNA isolation method. However, quantity and integrity of total RNA extracted with the TRI Reagent method were again significantly higher than with the alternate technique, confirming it as the superior method. The TRI Reagent isolation method also yielded high quality total RNA from white-tailed deer skin punch biopsies, suggesting the usefulness of this method for obtaining RNA of a quality suitable for gene expression studies in other ruminant species.