Asian isolates of Anaplasma phagocytophilum: Multilocus sequence typing

Anaplasma phagocytophilum is the bacterial agent of granulocytic anaplasmosis in humans and animals; it is widely distributed in Eurasia and North America and transmitted mainly by Ixodes ticks. Several approaches have been used to study genetic diversity in A. phagocytophilum, multilocus sequence typing (MLST) currently being the most reliable and comparable. The MLST method based on seven housekeeping loci, 2877 bp total length, has been used to create and maintain the MLST database available worldwide (https://pubmlst.org/aphagocytophilum/). Before this study, the database contained 150 sequence types (STs) and 418 isolates, 397 of them originating from Europe and 21 from the USA, with none from Asia. We typed 25 A. phagocytophilum isolated from Ixodes ticks collected in the Asian part of Russia and compared the results with the conventional 16S rRNA typing. Substantial variability in the primer binding sites was found, so we had to modify the original primers for six out of seven loci. None of the sequences obtained matched those from the database; 15 new STs and 39 new alleles were revealed. Russian isolates belonged to two clusters, cluster 1 (19 isolates) and 3 (6 isolates), in both of which they formed separate clades. For the first time, we found A. phagocytophilum isolates from Ixodes persulcatus and I. pavlovskyi to belong to cluster 3, previously containing only the strains from voles and shrews. Further research is needed to estimate the prevalence of two MLST clusters of A. phagocytophilum in ticks and vertebrate hosts in Asia.