Anaplasma phagocytophilum strains from voles and shrews exhibit specific ankA gene sequences
© Majazki et al.; licensee BioMed Central Ltd. 2013
Received: 10 May 2013
Accepted: 25 November 2013
Published: 28 November 2013
Anaplasma phagocytophilum is a Gram-negative bacterium that replicates obligate intracellularly in neutrophils. It is transmitted by Ixodes spp. ticks and causes acute febrile disease in humans, dogs, horses, cats, and livestock. Because A. phagocytophilum is not transmitted transovarially in Ixodes spp., it is thought to depend on reservoir hosts to complete its life cycle. In Europe, A. phagocytophilum was detected in roe deer, red deer, wild boars, and small mammals. In contrast to roe deer, red deer and wild boars have been considered as reservoir hosts for granulocytic anaplasmosis in humans, dogs, and horses according to groESL- and ankA-based genotyping. A. phagocytophilum variants infecting small mammals in Europe have not been characterized extensively to date.
We amplified the total ankA open reading frames of 27 strains from voles and shrews. The analysis revealed that they harboured A. phagocytophilum strains that belonged to a distinct newly described ankA gene cluster. Further, we provide evidence that the heterogeneity of ankA gene sequences might have arisen via recombination.
Based on ankA-based genotyping voles and shrews are unlikely reservoir hosts for granulocytic anaplasmosis in humans, dogs, horses, and livestock in Europe.
Anaplasma phagocytophilum is a Gram-negative bacterium that replicates obligate intracellularly in neutrophils . It is tick-transmitted and causes acute febrile disease in humans , in companion animals such as dogs , horses , and cats  as well as in livestock such as sheep and cattle [6, 7]. The main vector of A. phagocytophilum in Europe is Ixodes ricinus, whereas it is primarily transmitted by I. scapularis and I. pacificus in North America and by I. persulcatus in Asia .
Evidence exists that the naturally circulating A. phagocytophilum strains show a considerable degree of host adaptation, because they are not equally infectious for different animal species [3, 7–9]. The molecular characterization using major surface protein 2 (msp2) pseudogene sequences  as well as the ankA gene  has shown that strains originating from humans, dogs, and horses are homologous. Furthermore, horses and dogs are susceptible to infection with human A. phagocytophilum isolates [12–14].
At least in Ixodes spp. ticks A. phagocytophilum is not transmitted transovarially . Therefore, it is thought to depend on reservoir hosts to complete its life cycle. In North America, based on molecular characterization and experimental infections small mammals such as white-footed mice [16, 17], chipmunks [18, 19], and squirrels  were reported as probable reservoirs for granulocytic anaplasmosis in humans, horses, and dogs. In contrast, the impact of white-tailed deer and woodrats was questioned [18, 20, 21]. In Europe, A. phagocytophilum was detected amongst others in roe deer [22, 23], red deer , wild boars , hedgehogs , and other small mammals .
The 16S rRNA gene has been used most often for strain characterization. However, it was shown that it is not informative enough to delineate distinct A. phagocytophilum genotypes [11, 27–29]. Based on groESL and ankA gene sequences red deer [11, 30] and wild boar [31, 32] were considered as reservoir hosts for granulocytic anaplasmosis in humans, dogs, and horses. In contrast, roe deer harboured A. phagocytophilum strains which mostly belonged to clearly separated groESL and ankA gene clusters.
Apart from using the 16S rRNA gene the A. phagocytophilum variants infecting small mammals in Europe have not been typed extensively to date. We therefore amplified the total ankA open reading frame (ORF) of 27 strains from voles and shrews captured in Germany as well as the UK and compared them to 221 ankA sequences determined earlier [11, 27]. We here show that they harboured A. phagocytophilum strains that belonged to a distinct newly described ankA gene cluster. Therefore, voles and shrews are unlikely reservoir hosts for granulocytic anaplasmosis in humans, dogs, horses, and livestock in Europe.
Host species and geographic origin of A. phagocytophilum positive samples (n =34)
Canis lupus familiaris
Horse 32 FR
PCR analyses and sequencing
1 to 2 μl of DNA were used as template in a 50 μl reaction mixture containing 50 mM KCl, 20 mM Tris–HCl (pH 8.4), 2 mM MgCl2, 0.2 mM desoxynucleoside triphosphates, 0.4 μM of each primer, and 0.2 μl (1U) of Taq DNA Polymerase (Invitrogen, Karlsruhe, Germany). PCRs were performed using the GeneAmp PCR System 9700 (Applied Biosystems, Darmstadt, Germany) under the following conditions: initial denaturation at 94°C for 3 min, 40 cycles consisting of denaturation at 94°C for 30 s, annealing at the predicted melting temperature of the primers minus 4°C for 30 s, extension at 72°C for 30 s per amplification of 500 bp, and a final extension at 72°C for 10 min. Nested PCR amplification and sequencing of the A. phagocytophilum 16S rRNA gene [27, 38] and of the ankA gene clusters I  and IV  were performed as described previously. Nested PCR amplification and sequencing of the ankA gene cluster V was achieved as shown in Additional file 1: Table S1. The sequence of the complete ORF was obtained by assembling the sequences of the six nested PCR products. Nucleotide sequences of primers (Metabion, Martinsried, Germany) are summarized in Additional file 2: Table S2. Nested PCR products were directly sequenced bidirectionally using a 3130 Genetic Analyzer (Applied Biosystems) and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
Sequences were edited and assembled with the SeqMan program of the DNASTAR package (Lasergene, Madison, WI). For phylogenetic analysis of the 16S rRNA or ankA gene sequences the program MEGA 5.1  was used. Sequences were aligned by ClustalW applying the IUB matrix (16S rRNA gene) or codon-aligned applying the PAM (Dayhoff) matrix. Tree construction was achieved by the neighbor-joining method with the complete deletion option using the Jukes-Cantor matrix for nucleotide sequences and the PAM (Dayhoff) matrix for protein sequences, respectively. Bootstrap analysis was conducted with 1,000 replicates. Average distances within and net average distances between ankA gene clusters were computed using the same parameters as for tree construction. Protein sequences were analyzed for Pfam domain matches (http://pfam.sanger.ac.uk/) and for tyrosine kinase group phosphorylation sites (http://scansite.mit.edu/). Nucleotide consensus sequences were calculated for each ankA gene cluster with consensus maker v2.0.0 using the most common character and breaking ties with IUPAC characters (http://www.hiv.lanl.gov/content/sequence/HIV/HIVTools.html). The consensus sequences were codon-aligned by ClustalW applying the PAM (Dayhoff) matrix. The alignment was analyzed for recombination by Recco  with the Hamming mutation cost matrix and gap extension costs of 0.2. Events with seq p-values of < 0.5 and savings ≥ 5 were regarded as significant.
GenBank nucleotide accession numbers
Horse 32 FR
16S rRNA gene sequences
Seven of the 16S rRNA gene sequences from voles contained ambiguous nucleotides, indicating multiple infections with several 16S rRNA genotypes, a phenomenon that was observed already earlier in animal and tick samples [11, 27]. 14 of the 27 small mammals (11 voles and three shrews) harboured an A. phagocytophilum variant identical to [GenBank: M73220]. This genotype is widespread mainly in ruminants, but was also detected in voles and shrews [34, 35, 42]. Two 16S rRNA gene sequences were identical to [GenBank: AY082656] that was found in voles in the United Kingdom , whereas two matched [GenBank: GU236577] originating from red deer in Germany . Additionally, one vole was infected with an A. phagocytophilum variant identical to [GenBank: AY281785] and one with a new variant, respectively.
Net average identities* and similarities** between the different ankA gene clusters in percent
A search against the Pfam domain database demonstrated that all AnkA sequences from voles and shrews contained ankyrin repeats. Furthermore, multiple tyrosine phosphorylation sites were predicted by Scansite (http://scansite.mit.edu/) at their C-terminal end, one of them displaying a classical EPIYA motif . As described for AnkA clusters I and IV , the abundant tyrosine phosphorylation sites seemed to be arisen by duplication of direct repeats (Additional file 3: Figure S1).
In Europe, the reservoir hosts for A. phagocytophilum have not been clearly defined to date. The molecular characterization of A. phagocytophilum strains using groESL and ankA gene sequences revealed that red deer [11, 30] and wild boar [31, 32] might harbour variants that cause granulocytic anaplasmosis in humans, dogs, and horses. Small mammals were considered as reservoir hosts too, but it was shown that voles were infected with msp4 genotypes that differed from those of I. ricinus ticks . Because I. ricinus is the main vector of granulocytic anaplasmosis in humans and domestic animals in Europe , voles rather seem to be involved in a separate enzootic cycle probably with I. trianguliceps as tick vector . This is in line with our observation that voles and shrews harboured A. phagocytophilum strains that belonged to a newly defined distinct ankA gene cluster. Interestingly, we did not find sequences from I. ricinus ticks to cluster with those from voles and shrews supporting the hypothesis that A. phagocytophilum strains circulating in these small rodents are part of a completely separate ecology . Similarly, the groESL variants in voles and shrews from the Asian part of Russia were found to be clearly separated phylogenetically from all other analyzed strains . This is in contrast to the USA, where small rodents such as the white-footed mouse appear to be reservoir hosts for granulocytic anaplasmosis [16, 17]. Our results from the ankA-based phylogeny indicate that voles and shrews harbour A. phagocytophilum strains that might not be infectious for humans, dogs, horses, and livestock. However, other rodents species apart from those investigated here, could serve as reservoir hosts in Europe.
The AnkA protein is suggested to be secreted into host cells via the VirB/VirD-dependent type IV secretion system (T4SS) of A. phagocytophilum[45, 46]. After translocation it is tyrosine phosphorylated and thought to disturb host cell signalling via protein-protein interactions mediated by its ankyrin repeats [45, 46]. At its C-terminal end AnkA typically contains one classical EPIYA and multiple EPIYA-related motifs [11, 47] that undergo tyrosine phosphorylation . EPIYA motifs of bacterial effector proteins often show numerous duplications . We described this phenomenon before especially for AnkA clusters I and IV  and show here that this is also true for the AnkA cluster V associated with voles and shrews (Additional file 3: Figure S1).
For the effector protein CagA of Helicobacter pylori, it was shown that its EPIYA motifs expanded via point mutation and recombination . Our analysis of the five ankA consensus sequences revealed that the marked diversity of AnkA could have arisen via recombination as well (Figure 2). However, it was not possible to determine which sequences were the ancestral ones. It has been suggested that the diversification of EPIYA motifs may lead to altered or extended target-protein binding capacities . Therefore, a specific AnkA could mediate a distinct host tropism of a particular A. phagocytophilum isolate and be involved in host adapation. Accordingly, variability between strains from different host species was found mainly in the surface-exposed components of the T4SS of A. phagocytophilum.
If the ankA gene is indeed involved in host adaptation driven by recombination, the ankA-based phylogeny could be disturbed by the fact that one single recombination event can introduce multiple nucleotide exchanges at once. Therefore, other more conserved loci should be used to proof the phylogenetic separation of A. phagocytophilum strains from voles and shrews described here. Nevertheless, their marked dissimilarity to all other strains investigated, indicates a long evolutionary distance. As sequence data alone are not able to prove different biological strain properties, in vivo experiments should address whether A. phagocytophilum isolates from voles and shrews are infectious for humans, dogs, horses, and livestock.
Although there might be some sampling error in our data set, voles and shrews are unlikely reservoir hosts for granulocytic anaplasmosis in humans, dogs, horses, and livestock in Europe based on ankA genotyping.
Voles and shrews harbour A. phagocytophilum strains that contain ankA gene sequences belonging to the newly described cluster V that might have arisen via recombination. Because cluster V ankA sequences were restricted to voles and shrews, they are unlikely to serve as reservoir hosts for granulocytic anaplasmosis in humans, dogs, horses, and livestock in Europe.
For Germany, permission to trap rodents using snap traps was given by the District Government Stuttgart, Germany . For the United Kingdom, protocols for the handling and sampling of wild small mammals were approved by the University of Liverpool Committee on Research Ethics and were conducted in compliance with the terms and conditions of licenses awarded under the UK Government Animals (Scientific Procedures) Act, 1986 .
The samples of human and domestic animal origin were obtained as part of routine diagnostic evaluation. Informed consent was obtained from the patients and owners, respectively. Human samples 96 HE27 and 98 HE4 were kindly provided by Stephen J. Dumler (The Johns Hopkins School of Medicine, Baltimore, MD), human HGE-1  and dog Martin  samples by Ulrike G. Munderloh (University of Minnesota, St. Paul, MN), horse sample 32 FR by Daniel Schaarschmidt-Kiener (Laboratory at Zugersee, Hünenberg, Switzerland) and cow A262 and sheep F1480 samples by Martin Ganter (University of Veterinary Medicine, Hannover, Germany).
Availability of supporting data
All supporting data are included as additional files.
The authors are grateful to Jochen Maydt (London, United Kingdom) for his generous help with the recombination analysis. The article processing charge was funded by the German Research Foundation (DFG) and the Albert-Ludwigs-University Freiburg in the funding programme Open Access Publishing.
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