Background: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness in humans and animals. We used multilocus sequence typing (MLST) and ankA gene-based typing to study the molecular epidemiology of the A. phagocytophilum strains circulating in a German cattle herd over one pasture season. The aim was to investigate whether co-infection with two distinct variants, reinfection with the same and/or superinfection by a different strain occurred during one pasture season. Eight genetic loci were sequenced in 47 PCR-positive samples from 15 animals.
Results: Five different sequence types (ST) and four ankA alleles were detected in the cattle herd. Three different ST caused clinically overt tick-borne fever in primary infected animals. The concordance between ST and ankA allele was 100%. Therefore, the housekeeping genes used for MLST and the highly variable ankA gene were concatenated to increase resolution. Co-infection could be proven because samples of chronologically close collection dates were included. Co-infecting A. phagocytophilum strains differed by 14 to 18 single nucleotide polymorphisms (SNPs). Most superinfecting variants varied by 14 SNPs from the previous strain and appeared in median after a free interval of 31 days. Thus, it is unlikely that superinfecting strains arose by in-animal evolution. Immunity against re- or superinfection was assumed because the cattle developed clinical signs only during primary infection.
Conclusions: The tick-pathogen-vertebrate host interaction is probably much more complex than previously thought taking into account the frequently occurring events of co-infection, reinfection and superinfection. This complex situation could not be easily simulated in an experimental infection and underlines the value of field studies.
Keywords: Anaplasma phagocytophilum; Cattle; Germany; Multilocus sequence typing (MLST); Pathogenicity; Roe deer; Tick-borne fever; Variant; ankA.