Genotyping of Anaplasma phagocytophilum in natural endemic cycles

A. phagocytophilum is the causative agent for granulocytic anaplasmosis, which is a tick-transmitted emerging disease in humans and domestic animals. Genetic diversity in its genome could be a reason for a diverging pathogenicity in Europe compared to the USA. In order to maintain in nature, the bacterium has developed a survival strategy of different enzootic life cycles by circulating between hosts potentially developing clinical symptoms, reservoir hosts and ticks as vectors. In order to enhance the knowledge of possible endemic life cycles of A. phagocytophilum and its host-pathogen-associations, strains of A. phagocytophilum from different animal species were genetically characterized and phylogenetically classified on the basis of four partial genes (16S rRNA, groEL, msp4, msp2) within the present study. In total, 781 samples of 17 different animal species (dog, horse, cat, cattle, goat, hedgehog, red fox, roe deer, red deer, sika deer, fallow deer, mouflon, chamois, ibex, wild boar, bank vole, wood mouse), which were screened positive for A. phagocytophilum by real-time PCR, were considered in the present study. The animal samples included 425 samples already tested positive available from previous studies. Subsequently, all positive samples were investigated using conventional PCR and nested PCR protocols targeting the partial 16S rRNA, msp4, groEL and msp2 gene. Forward and reverse sequencing was performed with all amplified samples. The obtained nucleotide sequences were compared to each other and to corresponding sequences from the GenBank, including a phylogenetic analysis (neighbor joining method; bootstrap value: 1.000 repeats) of the groEL and the msp genes. Statistical analysis concentrated on the evaluation of diversity occurring in A. phagocytophilum of different animal species including the empirical variance of special animal groups, a trend analysis of the four analyzed genes and the odd`s ratio of common 16S rRNA variants. Altogether, 327 16S rRNA sequences, 172 groEL sequences, 174 msp4 sequences and 71 msp2 sequences from the present and previous studies were taken into consideration. The amplification of the partial 16S rRNA gene resulted in 23 variants, the groEL gene in 33 variants, the msp4 gene in 50 variants and the msp2 gene in 22 variants. A. phagocytophilum from wild cervids revealed a statistically higher mean and empirical variance of the analyzed genes in comparison to strains from domestic animals. Accordingly, domestic animals, red foxes and hedgehogs showed rather uniform nucleotide sequences of A. phagocytophilum. The calculation of the odd`s ratio of the most common 16S rRNA strains confirmed the preference of special variants in wild ruminants and domestic animal species, respectively. A preliminary genetic classification of the A. phagocytophilum strains into ruminant and non-ruminant variants was possible based on the partial 16S rRNA variants. The phylogenetic analysis of the groEL and the msp2 gene showed a clustering according to the three continents Europe, USA and Asia. On the contrary, the msp4 gene rather clustered strains according to host animal species of A. phagocytophilum, discriminating clusters with wild and domestic ruminant variants from clusters with human, dog and horse variants. Based on the genetic classification two endemic life cycles of A. phagocytophilum were proposed in the present study. The first life cycle includes hedgehogs, red foxes and red deer as reservoir hosts and domestic animals and humans as potential hosts developing clinical symptoms of the disease. The second endemic cycle of A. phagocytophilum possibly involves wild cervids and rodents as reservoir hosts and domestic ruminants as hosts. Since roe deer showed a high number of different variants of A. phagocytophilum, it might play a role in both suggested life cycles as reservoir host. However, the existence of an own subcycle of roe deer specific A. phagocytophilum strains is also possible.