Silencing expression of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections

Antimicrobial peptides, including defensins, are components of the innate immune system in ticks that have been shown to provide protection against both gram-negative and gram-positive bacteria. Varisin, one of the defensins identified in Dermacentor variabilis, was shown to be produced primarily in hemocytes but transcript levels were also expressed in midguts and other tick cells. In this research, we studied the role of varisin in the immunity of ticks to the gram-negative cattle pathogen, Anaplasma marginale. Expression of the varisin gene was silenced by RNA interference (RNAi) in which male ticks were injected with varisin dsRNA and then allowed to feed and acquire A. marginale infection on an experimentally-infected calf. Silencing expression of varisin in hemocytes, midguts and salivary glands was confirmed by real time RT-PCR. We expected that silencing of varisin would increase A. marginale infections in ticks, but the results demonstrated that bacterial numbers, as determined by an A. marginale msp4 quantitative PCR, were significantly reduced in the varisin-silenced ticks. Furthermore, colonies of A. marginale in ticks used for RNAi were morphologically abnormal from those seen in elution buffer injected control ticks. The colony shape was irregular and in some cases the A. marginale appeared to be free in the cytoplasm of midgut cells. Some ticks were found to be systemically infected with a microbe that may have been related to the silencing of varisin. This appears to be the first report of the silencing of expression of a defensin in ticks by RNAi that resulted in reduced A. marginale infections.This research was partially supported by the Oklahoma Agricultural Experiment Station (project 1669), the Walter R. Sitlington Endowed Chair for Food Animal Research (K. M. Kocan, Oklahoma State University), Pfizer Animal Health, Kalamazoo, MI, USA, the Junta de Comunidades de Castilla-La Mancha, Spain (project 06036-00 ICS-JCCM), and Ministry of Science and Education (MEC), Spain (project AGL2005-07401). Dr. Raúl Manzano-Roman was funded by Ministerio de Educación y Ciencia, Spain. V. Naranjo was founded by Consejería de Educación, JCCM, Spain. Support, in part, is gratefully acknowledged by a grant from the National Science Foundation, IBN 0212901 (Hynes & Sonenshine) and a grant from the National Research Fund for Tick-borne Diseases (Hynes & Sonenshine).Peer reviewe