Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction.
The aim of this study was to detect Anaplasma phagocytophilum in wild and domesticated animals and to identify the phylogenetic relationships of different strains of this bacterium. We adapted six published conventional methods targeting 16S fragments for real-time polymerase chain reaction. Initial screening of samples from 419 animals found 37 Anaplasma positives, later confirmed with several different primers and a TaqMan probe. We also performed DNA quantification and melting curve analysis. The nucleic acid of Anaplasma sp. was detected in a higher percentage of cases in members of the deer family, hares, bank voles and mice (12.5 approximately 15%) than in foxes, boars, cows, and horses (around 4 approximately 6%). We also performed blood analysis of cows, horses, mice, and ticks removed from animals, evaluating the presence of antibodies against granulocytic Anaplasma sp. Finally, we subjected 11 randomly selected PCR amplified products to direct sequencing and we constructed the corresponding phylogenetic tree with respect to the Ehrlichia equi sequence, homologous to the human granulocytic ehrlichiosis agent. Mutual identity of the sequencing ranged from 99% to 100%.
Hulínská
Dagmar
D
National Institute of Public Health, National Reference Laboratory for Lyme Disease, Division of Microbiology and Epidemiology, Prague, Czech Republic. dhulin@szu.cz
Langrová
Katerina
K
Pejcoch
Milan
M
Pavlásek
Ivan
I
D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification*; D041081:Anaplasma phagocytophilum / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D000829:Animals, Domestic / Q000382:microbiology*; D002417:Cattle; D017931:DNA Primers; D019084:Fluorescent Antibody Technique, Indirect; D006736:Horses; D051379:Mice; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity
D017931:DNA Primers