Segregation of genetic variants of Anaplasma phagocytophilum circulating among wild ruminants within a Bohemian forest (Czech Republic)

Abstract

Seroprevalence, infection rate, and genetic variants of Anaplasma phagocytophilum were studied in a synusia of large mammals (deer, moufflon, and wild boar) which occupied a natural, largely broad-leaved forest in Central Bohemia. Almost 250 animals were harvested during 1999–2002. Their serum and/or full blood as well as spleen tissues were sampled. The sera were tested for the presence of anti- A. phagocytophilum antibodies with IFA, and the blood and spleen DNA extracts were examined with nested PCR. Selected positive samples were sequenced in both directions using BigDye chemistry. Whenever a mixture was encountered, the PCR product was cloned, and several randomly selected clones were individually sequenced, to make the mixture's constituents clear. The established sequences were compared with each other, among host species, and also with the GenBank database. All 154 ruminant sera (moufflon, red deer, roe deer, fallow deer, and sika deer) showed reactive antibodies to A. phagocytophilum, with titres ranging from 1:320 to 1:10,240. By contrast, only 8 out of 34 (24%) wild boar sera showed elevated titres, from 1:80 to 1:1280. A. phagocytophilum DNA was detected in the blood samples ( n=66) of all ruminant species. The highest prevalence was shown in red deer (18/21) and the lowest in moufflon (1/28). The difference is statistically significant ( p=0.01). Two samples of wild boar blood tested negative, but one spleen sample was positive. The paired blood/spleen samples ( n=17) showed 71% correspondence in the PCR tests. Eleven extracts of DNA of A. phagocytophilum (6 red deer, 4 roe deer, 1 moufflon) were analysed in detail, and 4 genetic variants were distinguished based on partial 16S rRNA sequence. All red deer showed dual infections with a variant already reported from this host (DV), accompanied by a variant which was undistinguishable from that associated with horse, dog, and human infections in Europe (HV). All the roe deer hosted a variant peculiar to that species (RV); this variant was either found singly or mixed with HV. The unique moufflon isolate corresponded to a variant reported from sheep in Europe (SV). Any genetic variant, with the exception of HV, was only detected within a single ruminant species. Fisher's exact test proved that the association of RV and DV with the respective roe and red deer is statistically significant ( p=0.005). This study indicates that while circulating in nature, the variants of A. phagocytophilum in wild ruminants tend to be segregated from one another by species barriers despite the close relationships of their hosts, their sympatric ranges as well as a shared population of vector ticks.