Development of nonradioactive in situ hybridization for detection of Anaplasma marginale in ticks

In situ hybridization (ISH), which allows localization of pathogen nucleic acid in tissue sections, was used to detect Anaplasma marginale, a rickettsial pathogen of cattle, in its tick vector. Dermacentor andersoni male ticks were experimentally infected with A. marginale, and one-half of each of 20 ticks was embedded in paraffin or LR White for ISH: Companion halves were embedded in DER resin for light microscopy (LM). Sections were digested with proteinase K and hybridized with a digoxigenin-labeled DNA probe. In both paraffin and LR White embedded sections, dark-blue color precipitates of hybridization signals were visualized in both salivary gland and gut cells. Anaplasma infections were also confirmed by LM in companion tick halves. Positive hybridization signals and A. marginale colonies were not seen in uninfected control tissues. Although ISH detected A. marginale in both paraffin and LR White embedded sections, LR White was found to be optimum for ISH of A. marginale because of improved morphological perservation and the superior resolution of the 1 mu m sections, allowing definitive identification in tick tissues.