Nl. Ge et al., DEVELOPMENT OF NONRADIOACTIVE IN-SITU HYBRIDIZATION FOR DETECTION OF ANAPLASMA-MARGINALE IN TICKS, Journal of histotechnology, 20(2), 1997, pp. 103-108
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.