DEVELOPMENT OF NONRADIOACTIVE IN-SITU HYBRIDIZATION FOR DETECTION OF ANAPLASMA-MARGINALE IN TICKS

In situ hybridization (ISH), which allows localization of pathogen nuc leic acid in tissue sections, was used to detect Anaplasma marginale, a rickettsial pathogen of cattle, in its tick vector. Dermacentor ande rsoni male ticks were experimentally infected with A. marginale, and o ne-half of each of 20 ticks was embedded in paraffin or LR White for I SH: Companion halves were embedded in DER resin for light microscopy ( LM). Sections were digested with proteinase K and hybridized with a di goxigenin-labeled DNA probe. In both paraffin and LR White embedded se ctions, dark-blue color precipitates of hybridization signals were vis ualized in both salivary gland and gut cells. Anaplasma infections wer e also confirmed by LM in companion tick halves. Positive hybridizatio n signals and A. marginale colonies were not seen in uninfected contro l 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 sup erior resolution of the 1 mu m sections, allowing definitive identific ation in tick tissues.