Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction.

This research study aims to identify Anaplasma phagocytophilum, a bacterium, in a range of wild and domestic animals using advanced testing and analysis techniques. The prevalence of this bacterium was also assessed in different animal groups and DNA samples from positive tests were used to understand the genetic relationships of different strains of the bacterium in relation to a known infectious agent.

Methods and Initial Findings

• The researchers adapted six existing methods targeting 16S fragments for real-time polymerase chain reaction (PCR), a technique used to amplify and quantify DNA sequences.
• 419 animals were screened for the presence of Anaplasma using these methods, with 37 initially testing positive.
• The positive results were confirmed with various primers and a special type of genetic probe called a TaqMan probe.
• In addition to detection, they performed DNA quantification and melting curve analysis, which measures how the DNA changes with varying temperatures.

Prevalence in Different Animals

• Analysis revealed that the detection rate of the Anaplasma bacterium was higher in certain animals–specifically, members of the deer family, hares, bank voles, and mice, where the detection rate ranged from 12.5% to 15%.
• In comparison, lower detection rates (around 4% to 6%) were seen in foxes, boars, cows, and horses.

Blood Analysis and Antibody Detection

• The researchers also performed blood tests on cows, horses, mice, and ticks taken from animals to investigate the presence of antibodies against granulocytic Anaplasma sp., which could indicate a past or present immune response to the bacterium.

DNA Sequencing and Phylogenetic Analysis

• 11 random PCR products were selected for direct sequencing to identify and investigate the bacterial DNA.
• The sequencing data were used to create a phylogenetic tree, a diagram that shows the evolutionary relationships among various biological species or other entities.
• The phylogenetic tree was compared to the Ehrlichia equi sequence, a sequence similar to the human granulocytic ehrlichiosis agent, to understand the genetic relationships between the bacteria.
• The identities of the sequences ranged from 99% to 100%, indicating a high degree of genetic similarity.

This study provides valuable insights into the detection and prevalence of Anaplasma phagocytophilum in wild and domesticated animals, and the genetic relationships of different strains of this bacterium.