Development of a Non‐radioactive DNA Probe andin SituHybridization for Detection ofAnaplasma marginalein Ticks and Cattle^a

Abstract

A non-radioactive DNA probe was developed for detection of Anaplasma marginale in ticks and cattle. The probe was labeled with digoxigenin 11-dUTP by polymerase chain reaction. The probe was tested on bovine blood and was found to be a sensitive and specific detection method for A. marginale in cattle. The DNA probe was then adapted for in situ hybridization (ISH) of A. marginale in Dermacentor andersoni and D. variabilis ticks infected either as nymphs or adults. One-half of each tick was studied with ISH while the other half was examined with light and electron microscopy. In male ticks infected as adults, tick gut cells first became infected with A. marginale while ticks fed on an infected calf, and they remained infected as they transmission fed on a second, susceptible calf. At the onset of transmission feeding, salivary glands became infected with A. marginale. During transmission feeding infection was also observed in interstitial, reproductive, skeletal muscle, fat body and Malpighian tubule tissue, resulting in a generalized A. marginale infection. When adult ticks that acquired infection as nymphs were examined with ISH and microscopy, gut tissues of both D. andersoni and D. variabilis became infected with A. marginale. However, salivary gland infection was seen only in D. variabilis, even though both species of ticks transmitted A. marginale to susceptible calves. A. marginale was not seen with ISH or microscopy in hemocytes collected from both species of ticks and, thus, hemocytes do not appear to play a role in the development of A. marginale in ticks.

Differential Expression and Sequence Conservation of theAnaplasma marginale msp2Gene Superfamily Outer Membrane Proteins

“…Whether a functional omp6 accumulated mutations and deletions, resulting in pseudogene formation, or was never functional is unknown. There are also two alternative explanations for the lack of detectable transcription and expression for omp2, omp3, and omp6: (i) these omp genes are expressed only at very low levels, below the sensitivity of detection for both assays, or (ii) expression is tightly restricted and occurs only in tissues other than those tested, as infection has been detected in tissues other than tick midguts and salivary glands (18,21). Regardless, as the tick midgut and salivary glands and bovine erythrocytes represent the key sites for invasion, replication, and maturation in the transmission cycle, and given the low level of expression, if any, the data suggest that OMP2, OMP3, and OMP6 are unlikely to be involved in the key steps of transmission.…”