BACKGROUND
the dramatically reducing cost of high-throughput sequencing  technologies has enabled their use across a wide range of bacterial genome sequencing projects  <cit> .
hts can now even be used for routine medical investigations in bacteriology  <cit> .
to effectively perform whole genome sequencing  on obligate intracellular bacteria, microorganism isolation, culture and dna purification are often essential steps.
the difficulty, or even impossibility, of cultivating some of these bacteria, can be a critical barrier to accessing their genomic sequences.
some authors attempted to sequence the genomes of intracellular bacteria without culturing steps, but successful approaches seem to be rare  <cit> .
such difficulties result in an underutilization of hts technologies when studying this type of bacteria.
anaplasma phagocytophilum is a tick-borne alpha-proteobacterium  <cit> .
it infects a large range of hosts, including humans, wild and domestic ruminants, dogs, horses, and rodents  <cit> .
this bacterium is challenging to isolate and cultivate, as it replicates in short lifespan cells , which rapidly undergo autolysis after sampling.
for this reason, only nine a. phagocytophilum genomes are currently available, of which just three are complete  <cit> .
apart from norway variant  <dig>  obtained from a norwegian sheep, all genomes correspond to north american strains: human strains hz, hz <dig>  and hge <dig>  dog <dig> dog strain, mrk horse strain, jm rodent strain, and the tick  strains crt <dig> and crt <dig>  a. phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, horses, dogs and occasionally cats, and tick-borne fever  in domestic ruminants  <cit>
.
however, the epidemiology of a. phagocytophilum infection differs greatly between europe and the usa.
in the usa, human granulocytic anaplasmosis  is an increasing public health problem, with a five-fold increase in the number of cases between  <dig> and  <dig>  <cit> , whereas no tbf cases have been described to date.
in contrast, hga appears to be rare in europe , whereas tbf cases are severe in cattle and sheep, causing significant economic losses .
these different epidemiological contexts are associated with considerable strain variations  <cit> .
an american strain infectious for horses is not infectious for ruminants  <cit> , while a european variant pathogenic for cattle does not cause any clinical disease in horses  <cit> .
in the usa, the ap-variant  <dig> infects goats and deer, but not mice , whereas the ap-ha variant can infect both ruminants and mice under experimental conditions  <cit> .
taken together, these results suggest that distinct a. phagocytophilum ecotypes with varying host tropisms, circulate in europe and the usa.
the genetic diversity of a. phagocytophilum must be explored in order to investigate its phylogeny, and to also identify genetic markers capable of distinguishing ecotypes.
for this purpose, genome sequences from various animal strains are needed.
additionally, given the significant economic consequences of tbf infection in europe, particularly in france, it is important to specifically focus on bovine a. phagocytophilum.
within this context, our objective was to sequence and characterize the genome of a. phagocytophilum  obtained from a cow  with tbf, and generate comparisons with the other available a. phagocytophilum genomes.
therefore we followed a whole genome solid-phase sequence capture approach, which allowed us to sequence the genome of a cow sample without the need for strain isolation.
this strategy has already been successfully used to sequence the genome of the arthropod symbiotic bacterium wolbachia, another obligate intracellular microorganism  <cit> .
to the best of our knowledge, our study is the first to provide the genome sequence of a bovine strain.
following comparison with nine available genomes, we then identified core and accessory a. phagocytophilum genes.
core genes comprised the set of orthologous genes shared among the ten sequenced a. phagocytophilum genomes, whereas accessory genes were those orthologous genes not shared among the ten genomes.
four genes were specific to the a. phagocytophilum bovine genome, and nine were common to both genomes from domestic ruminants .
as all of these genes code for “proteins of unknown function without similarity to other proteins” their functions must now be explored.
RESULTS
confirmation of a. phagocytophiluminfection
in february  <dig>  one cow  was diagnosed with tbf at the national veterinary school of toulouse .
a. phagocytophilum infection was confirmed by the observation of morulas in blood smears  and by msp <dig> pcr amplification.
another cow sample , was confirmed as uninfected by a. phagocytophilum , and was used as a negative control.figure  <dig>
morulas observed in the neutrophils of the bovine blood sample 10_ <dig>
whole genome capture and sequencing of a. phagocytophilum
the whole genome capture step was performed using a nimblegen solid-phase capture array which encompassed  <dig> ,085 bp, corresponding to  <dig> % of the hz reference strain genome.
the library thus obtained was sequenced using the illumina hiseq  <dig> platform with a paired-end protocol, and a total of  <dig> , <dig> reads were retrieved.
subsequently, these reads were aligned to a. phagocytophilum and cattle genomes.
a total of  <dig> , <dig> properly paired reads mapped to the pathogen genome, corresponding to  <dig> % of the total number of reads.
this percentage is similar to that obtained by other groups, such as by bright et al.
<cit> .
on the other hand,  <dig> , <dig> properly paired reads  were unmapped.
after the alignment step, the reads mapping to the cattle genome were discarded and not considered for subsequent analysis.
for the subset of reads which mapped to the pathogen genome, further pcr duplicate detection was performed.
pcr-duplicated reads are known to arise during the final amplification steps of library preparation, and are related to the reduced complexity of captured libraries.
in our case,  <dig> , <dig> duplicated reads were found in the dataset mapping to a. phagocytophilum, which corresponded to a percentage of  <dig> %.
this value is relatively high as compared to those obtained by other authors in similar contexts.
for instance, bright et al.
reported to have obtained percentages of duplicated reads ranging from  <dig> % to  <dig> % in plasmodium vivax field samples, with an average of  <dig> %  <cit> .
the properly paired a. phagocytophilum reads retained after the removal of duplicates  were combined with the properly paired unmapped reads  to obtain a final dataset of  <dig> , <dig> reads, roughly corresponding to  <dig>  gb of data and representing a sequencing depth of  <dig> x. due to the massive amount of raw data, the reads were first digitally normalized using khmer  <cit> .
subsequently,  <dig> , <dig> properly paired reads were retained  and then de novo assembled using velvet.
the assembly resulted in  <dig> scaffolds  with lengths of 1 kb or more.
the n <dig> value was  <dig>  bp with a maximum length of  <dig>  kb and a minimum length of  <dig>  bp.
the total length of the assembly was  <dig> , <dig> nucleotides, consistent with the sizes of other a. phagocytophilum genomes.
after the assembly of the bov-10_ <dig> genome,  <dig> % of the sequences included in the scaffolds aligned to the hz genome .
the  <dig> % remaining sequences within the scaffolds did not map to the genome.
these regions could either be absent from the hz genome and/or too variable to correctly align with the hz genome .
on the other hand, only  <dig>  % of the hz genome sequence aligned to bov-10_ <dig> scaffolds.
for the remaining hz regions it was not possible to determine if they were indeed absent from the bov-10_ <dig> genome, or if they were missing due to biases introduced during the amplification steps  or because of biases related to the capture steps .
using bov 10_ <dig>  new capture probes could be designed, which should improve the capture rate of a. phagocytophilum dna in bovine field samples.figure  <dig>
dot plot of assembled scaffolds versus hz genome.
dot plot of contigs  <dig> versus hz genome.
in order to calculate bacterial dna fold-enrichment, we estimated the amount of both a. phagocytophilum dna mass and bovine dna mass in sample 10_ <dig> using six different qpcr amplifications: three which targeted a. phagocytophilum genes and three which targeted bovine genes .
the relative abundance of the a. phagocytophilum dna within the original 10_ <dig> sample corresponded to approximately  <dig> % of the total dna by mass .
since the relative abundance was approximately 59% after the whole genome capture step, the level of enrichment was 197-fold, a rather high value.
in fact, results obtained from other similar technical contexts appear to range from ~20-fold to ~100-fold  <cit> , and in some cases the level of variability among samples was significant  <cit> .
the value obtained in this study could be explained by considering that in our protocol we used 300 μg of cot- <dig> bovine dna during the hybridization step, instead of 100 μg as suggested by the nimblegen standard method.
this modification could have increased the level of capture specificity by reducing non-specific hybridization.table  <dig>
genes targeted and primers used for quantification of bovine and
a. phagocytophilum
dna
a. phagocytophilum
anka
glta
groel
bos taurus
gapdh
ywhaz
ppia
a. phagocytophilum
dna within the original 10_ <dig> sample
anka
groel
glta
as previously stated, we obtained approximately 986x genome coverage, an extremely high value, much higher than that required to achieve a whole genome assembly of high quality .
such extensive coverage was most likely generated during the experimental planning stage, as we were unable to predict the enrichment level achievable with an initial starting concentration of only  <dig> % a. phagocytophilum dna.
therefore, to ensure that the read number would be sufficient, we used an entire hiseq  <dig> lane for the sequencing.
this first experience shows that it should be relatively simple to sequence several a. phagocytophilum genomes via multiplexing, without compromising the final results in terms of coverage.
the only possible issue is that the enrichment ratio could in fact be rather variable from sample to sample, as observed by bright et al.
<cit> .
in conclusion, we have successfully demonstrated the effectiveness of a whole genome capture approach to selectively enrich for pathogenic bacterial dna that was originally mixed with host mammalian dna.
this is the first report where an a. phagocytophilum genome sequence has been obtained without any culturing steps.
as previously stated, whole genome capture has already been used to sequence the genome of the arthropod non-pathogenic symbiont wolbachia, another obligate intracellular bacterium  <cit> .
here, we demonstrate that whole genome capture is also a suitable approach for pathogenic bacteria wgs such as a. phagocytophilum, and that it could be adapted for large-scale studies of these bacterial genomes, directly from field samples.
features of a. phagocytophilumgenomes
a. phagocytophilum has a single circular chromosome, without any identified plasmids  <cit> .
the main characteristics of the ten genomes investigated in this study are summarized in table  <dig>  for bov-10_ <dig>  the overall gc content was  <dig> % as for other a. phagocytophilum genomes.
this value is very high compared to other rickettsiales-order bacteria, as the genome decay of these bacteria is often accompanied by a low gc% content .
a total of  <dig> cds  were predicted, of which  <dig> were complete.
in addition,  <dig> rrna and  <dig> trna coding sequences were also identified.
overall, genome size appeared to be conserved among the ten strains , whereas the number of genes varied greatly .
these differences could be explained, at least in part, by fragmentation of bov 10_ <dig> incomplete sequence assemblies.
over 70%  of the cds had an assigned function.table  <dig>
characteristics of the ten
a. phagocytophilum
genomes investigated in this study
*genes include complete cds, rrna, trna and pseudogenes.
phylogenetic analysis
phylogenetic trees based on entire sequences of groel, glta, msp <dig> and 16s rrna loci were constructed .
the two european ruminant strains clustered together in all four phylogenetic trees.
the crt <dig> and crt <dig> ixodes scapularis strains were also associated within the four phylogenic trees, whereas the three human strains, the jm rodent strain and the dog <dig> dog strain were always grouped together in the same separate cluster.
finally, the mrk horse strain clustered with the human strains only in the cases of 16s rrna and msp <dig> phylogenic trees.figure  <dig>
phylogenetic trees based on the
groel
,
glta
, 16s rrna  and
msp4
loci.
analyses were performed by the neighbor-joining method using  <dig> bootstraps.
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test  are shown next to the branches.
comparison of the gene content of other a. phagocytophilumgenomes
in order to compare gene content of the ten genomes investigated in this study, we first identified their core and accessory genomes by defining ortholog clusters at the protein level .
following analysis,  <dig> clusters were obtained,  <dig>  of which belonged to the core genome .
bov-10_ <dig> shared from between  <dig> to  <dig> proteins with the other nine strains considered in this study .
as for other anaplasmataceae, in which the vast majority of genes belonged to the core genome , we were surprised to observe that a. phagocytophilum contained an exceedingly small proportion of such core genes.
this could be due, at least in part, to the application of various annotation methods used for the ten studied genomes , leading to differing gene predictions.
in addition, it should be mentioned that  only the hz, hz <dig> and jm published genomes are fully sequenced , and  the genome annotation of the hz strain is of higher quality in comparison to the others, because proteomic data were also used  <cit> .
moreover,  <dig> clusters were found only in one strain .
a large number of these clusters  corresponded to the msp2/p <dig> gene family.
this is not surprising, as these genes tend to have hypervariable central regions, therefore such low global similarity levels prevent their assembly into one cluster.
altogether, these factors could have led to a significant underestimation of genes belonging to the core genome.
however, nine gene clusters were only detected in the genomes of two european domestic ruminant strains, and four additional clusters were unique to the genome from our bovine sample, when compared to available american a. phagocytophilum genomes .
as all corresponding proteins were annotated as "hypothetical proteins", their functions have yet to be explored.
for instance, the proteins corresponding to the  <dig> aforementioned gene clusters could be involved in host tropism for those strains that infect domestic ruminants and/or could be specific to the european strains.
in order to examine whether the differences between the european strains are related to animal species or to geographical location, the presence of these genes in a. phagocytophilum genomes from different wild and domestic animals living in the same areas should be investigated.
the strategy developed in this paper would be an effective method to rapidly sequence these genomes.figure  <dig>
diagram of the number of proteins shared by different
a. phagocytophilum
strains.
number of proteins shared between bov-10_ <dig> and the nine other strains considered in this study
small peptides
genes encoding small peptides  are common features of both prokaryote and eukaryote genomes, and are involved in many biological functions  <cit> .
for staphylococcus aureus, one small peptide class is involved in both virulence and modulation of host immune responses  <cit> .
in bov-10_ <dig>  we detected  <dig> cds  which coded for small peptides.
all predicted small peptides had either no known function or were designated as p44/msp <dig> pseudogenes.
in total, 31%  of predicted cds in the hz genome coded for small peptides  <cit> .
of these small peptide coding sequences, 67%  are actually produced  <cit> , confirming that the majority are not only due to false positive gene prediction during genome annotation.
however 82%  of the expressed small peptides have unknown functions  <cit> .
in order to better understand the biology of a. phagocytophilum, the functions of these small peptides must be explored.
adhesion and host cell internalization genes
both the adhesion to and internalization of a. phagocytophilum within host cells are mediated by multiple bacterial adhesins/invasins that cooperatively recognize host cell receptors.
to date, at least six a. phagocytophilum adhesins/invasins have been identified.
these six proteins belong to the core genome of the ten strains assessed in this study.
ompa binds to α <dig> -sialic acid of the sialyl lewis x-tetrasaccharide that caps p-selectin glycoprotein ligand- <dig> , and msp <dig> appears to recognize α- <dig> -fucose and/or psgl-1 n-terminal peptide  <cit> .
asp <dig> and aipa both recognize an unknown receptor  <cit> .
a. phagocytophilum adhesion could also involve asp <dig> and asp <dig>  two other a. phagocytophilum surface proteins, as indicated by neutralization studies  <cit> .
in bov-10_ <dig> and crt <dig> strain genomes, we detected a 14-amino-acid-deletion in the n-terminal region of asp <dig>  the asp <dig> domain essential for cellular adherence and invasion is located between the  <dig> to  <dig> c-terminal amino acids   <cit> , therefore this deletion is not expected to have any effect on bacterial adhesion.
interestingly, we detected one substitution at position  <dig>  which was only observed in ruminant strains.
the ompa protein is completely conserved between strains, except for the domestic ruminant variants; norway variant  <dig> and 10_ <dig>  in whom five substitutions were detected.
however, as these substitutions lie outside the essential binding domain of ompa  <cit> , they are also not expected to have any effect on bacterial adhesion.
in the hz strain, aipa is a  <dig> amino acid protein, however in crt <dig>  dog <dig>  jm, mrk and hz <dig> strains, we detected a deletion of  <dig> amino acids in the aipa n-terminal region.
in strains 10_ <dig>  crt <dig> and hge <dig>  we detected an additional deletion of four amino acids in the same n-terminal region.
however a nucleotide blast search of the hz aipa nucleic sequence revealed that this gene is actually intact in the ten studied genomes.
the start codon for this gene is “ttg, therefore this inaccuracy could be due to difficulties with start codon detection during genome annotation.
we also detected  <dig> deletions and substitutions in aipa proteins across the ten strains.
further analyses are required in order to draw conclusions on the functional impact of these variations.
the hz genome contains one msp <dig> locus, two msp <dig> homolog loci, and  <dig> p <dig> loci  <cit> .
in order to evade the host immune system, a. phagocytophilum utilizes gene conversion to shuffle msp <dig> pseudogenes into the single msp <dig> gene expression cassette .
one proteomic study has shown that in addition to the expression site aph_ <dig>  full-length p <dig> genes can be expressed at their own loci, whereas silent p <dig> genes have to be recombined into the expression site for protein production  <cit> .
the locus  expressed during the interaction of msp <dig> with psgl- <dig> is/are not known.
thus it was not possible to further investigate the role of msp2/p <dig> variations in a. phagocytophilum binding.
moreover the binding sequence on msp <dig> remains undefined.
asp <dig> and asp <dig> are the least conserved proteins across all the strains, containing 58/ <dig>  and 99/ <dig> amino acid substitutions or deletions, respectively.
however protein characterization studies must still be performed to determine the functional impact of these variations.
genes encoding proteins involved in secretion systems
protein export systems are extremely important for host-pathogen interactions.
gram-negative bacteria often contain the general secretion  and the twin-arginine translocation  pathways, both involved in exporting protein into the periplasm; and specialized export systems, such as the type iv secretion system, dedicated to exporting specific subsets of proteins .
all ten strains studied in this work contained both the sec and tat pathways, as well as type i and type iv specialized export systems.
sec pathway
all strains contained eight genes  of the sec pathway .
detection of potential sec substrates was performed as described in the material and methods section, and are listed in additional file  <dig>  many proteins detected as potential sec substrates are surface outer membrane proteins .
these proteins may be secreted into the bacterial periplasm by the sec pathway, and then anchored in the outer membrane.
the virb <dig>  virb <dig> and virb <dig> subunits of the type iv secretion system have also been detected as potential sec substrates.
virb <dig> and virb <dig> proteins are periplasmic and/or outer membrane channel subunits of the t4ss, and virb <dig> could be surface exposed in a. phagocytophilum
<cit> .
the sec pathway may secrete these proteins into the bacterial periplasm for assembling, whereas other vir proteins are localized to the inner membrane of a. phagocytophilum, and do not require secretion.
seven proteins  involved in various cellular processes such as cell metabolism, and two lipoproteins  with unknown localization, were also detected as potential sec substrates.
ten hypothetical proteins of unknown function were also identified.
these proteins could be uncharacterized outer membrane proteins, or might perform their biological activity in periplasmic compartments; consequently their functions need to be explored.
the majority of the proteins considered as potential sec substrates were detected in all ten genomes.
however some of these proteins were only detected in some of the compared genomes, such as crt38_ <dig> which was only present in the crt <dig> and crt <dig> genomes .
tat pathway
the tat pathway is found in most bacteria and has been proven to be essential for virulence in several pathogens  <cit> .
in escherichia coli and anaplasma marginale, the minimal set of genes required for tat translocation and a functioning tat system consists of tata, tatb, and tatc
<cit> .
all strains examined in this study contained tata, tatb, and tatc, suggesting that they produce functional tat pathways.
it is interesting to note that while tat proteins are not synthesized during hl- <dig> cell infection  <cit> , tata is expressed in tick salivary glands, suggesting that a. phagocytophilum may utilize the tat pathway during tick infection  <cit> .
contrary to many other α-proteobacteria, but consistent across rickettsiae, the three tat genes are dispersed throughout the genome  <cit> .
our in silico investigations led to the prediction of two potential tat substrates across the ten a. phagocytophilum strains.
the first had 33% and 28% amino acid identity with the omla protein of azospirillum sp and wolbachia wno, respectively.
omla is an outer membrane lipoprotein involved in the maintenance of cell envelop integrity in pseudomonas aeruginosa
<cit> , and possibly virulence in xanthomonas axonopodis
<cit> .
as lipoproteins are particularly immunogenic and often have adjuvant activity, omla could be a useful vaccine candidate for pathogenic bacteria.
as such, it has already been used in pigs to protect against actinobacillus pleuropneumoniae
<cit> .
the fact that omla is present in all the a. phagocytophilum strains studied here, reinforces its potential efficacy as a vaccine candidate against this bacterium.
however, this protein is not conserved across all strains.
for example, a deletion of approximately  <dig> amino acids, containing a cysteine residue involved in lipid attachment, is observed in the omla n-terminal region of the hz strain.
this observation suggests that omla may not be a surface-exposed protein in the hz strain, raising doubts about the utility of this protein as a vaccine candidate.
the second potential tat substrate is a ubiquinol-cytochrome c reductase iron-sulfur subunit involved in the electron transport chain.
to date, both the tat and sec pathways have been poorly studied in a. phagocytophilum.
due to their importance in other pathogenic bacteria, further investigations are needed in order to fully explore their specific roles in a. phagocytophilum virulence.
t1ss secretion system
the type i secretion system  spans the periplasm and both inner and outer membranes of gram-negative bacteria, and facilitates protein secretion across these compartments.
the t1ss is composed of three major proteins: atp-binding cassette  transporters, outer membrane factors , and membrane fusion proteins   <cit> .
all ten strains in this study contained these three protein-encoding genes.
it remains to be elucidated whether these genes code for an efficient t1ss.
however, the strong conservation of the genes encoding this secretion system that has evolved through reductive evolution, observed across all ten a. phagocytophilum genomes,  <cit> , suggests its importance in the bacterial lifecycle.
many t1ss substrates are involved in bacterial virulence .
in ehrlichia chaffeensis, three secreted proteins containing tandem repeats and the ankyrin repeat protein, ank <dig>  involved in host pathogen interactions, are t1ss substrates  <cit> .
to date, no t1ss substrate has been described in anaplasma spp.
identifying tss <dig> substrates may be a significant step towards a better understanding of a. phagocytophilum biology.
t4ss secretion system
the type iv secretion system  is a multi-protein complex that also spans the periplasm and both membranes of gram-negative bacteria.
it can also span eukaryotic host cell membranes and has the ability to transport both nucleic acids and proteins into eukaryotic host cells, and to interfere with host signaling.
the t4ss is essential for the survival and virulence of many intracellular bacteria  <cit> , and the structure of the a. phagocytophilum t4ss has been well studied  <cit> .
each strain studied here contained one copy of virb <dig>  virb <dig>  virb <dig>  and vird4; two copies of virb <dig> ; virb <dig> and virb9; four copies of virb <dig> ; and a variable number of virb <dig> homologue genes.
virb <dig> proteins constitute the secretion channel and are the most diverse of the t4ss subunits in a. phagocytophilum, both in terms of copy number and sequence  <cit> .
we found  <dig> homologue virb <dig> genes in the hge <dig> genome,  <dig> in hz, hz <dig>  and 10_ <dig> genomes,  <dig> in dog <dig>  jm, mrk, and norway variant  <dig> genomes,  <dig> in the crt <dig> genome, and  <dig> in the crt <dig> genomes.
as already described in previous studies, we found that all strains analyzed here lacked both virb <dig> and virb <dig> genes  <cit> .
the role of t4ss in a. phagocytophilum virulence has been well studied.
two a. phagocytophilum t4ss substrates are currently recognized: i) the ankyrin repeat domain-containing protein a , which interacts with host proteins and dna , ii) the anaplasma translocated substrate  <dig> , involved in the induction of autophagosome formation and potential interference with apoptosis induction .
the ats- <dig> gene was detected in all ten genomes in this study, whereas anka was absent from the crt <dig> genome.
as the crt <dig> genome was composed of two contigs, separated by a gap, we cannot conclude if anka was really absent from this genome, or if the gene was not sequenced or assembled because it happened to fall within the gap.
other secretion systems
genes representing components of other secretion systems  were not detected in any of the genomes analyzed here, as previously described for the hz genome  <cit> .
however, some proteins, such as aph_ <dig>  which lack a secretion signal, have been detected on the bacterial surface, indicating that a. phagocytophilum may have alternative motifs directing proteins to cell surface  <cit> .
CONCLUSIONS
in this study we have used an innovative approach in order to sequence the genome of a bovine sample of a. phagocytophilum, and compare it to other published genomes.
whole genome capture has already been successfully used for wolbachia genome studies  <cit> .
here we demonstrated that this approach is also applicable to a pathogenic bacterium, a. phagocytophilum, and permits the sequencing of whole genomes without any need for strain isolation.
for this reason, we strongly believe that our approach generates a promising tool for large-scale studies of a. phagocytophilum genomes, directly from field samples.
genome comparison allowed us to identify four proteins specific to the a. phagocytophilum bovine genome, and nine proteins specific to the two available european domestic ruminant strains.
as these proteins could be involved in ruminant strain host tropism, their functions necessitate further exploration.

 the dramatically reducing cost of high-throughput sequencing technologies has enabled their use across a wide range of bacterial genome sequencing projects. hts can now even be used for routine medical investigations in bacteriology. to effectively perform whole genome sequencing on obligate intracellular bacteria, microorganism isolation, culture and dna purification are often essential steps. the difficulty, or even impossibility, of cultivating some of these bacteria, can be a critical barrier to accessing their genomic sequences. some authors attempted to sequence the genomes of intracellular bacteria without culturing steps, but successful approaches seem to be rare. such difficulties result in an underutilization of hts technologies when studying this type of bacteria. anaplasma phagocytophilum is a tick-borne alpha-proteobacterium. it infects a large range of hosts, including humans, wild and domestic ruminants, dogs, horses, and rodents. this bacterium is challenging to isolate and cultivate, as it replicates in short lifespan cells, which rapidly undergo autolysis after sampling. for this reason, only nine a. phagocytophilum genomes are currently available, of which just three are complete. apart from norway variant obtained from a norwegian sheep, all genomes correspond to north american strains: human strains hz, hz and hge dog dog strain, mrk horse strain, jm rodent strain, and the tick strains crt and crt a. phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, horses, dogs and occasionally cats, and tick-borne fever in domestic ruminants. however, the epidemiology of a. phagocytophilum infection differs greatly between europe and the usa. in the usa, human granulocytic anaplasmosis is an increasing public health problem, with a five-fold increase in the number of cases between and, whereas no tbf cases have been described to date. in contrast, hga appears to be rare in europe, whereas tbf cases are severe in cattle and sheep, causing significant economic losses. these different epidemiological contexts are associated with considerable strain variations. an american strain infectious for horses is not infectious for ruminants, while a european variant pathogenic for cattle does not cause any clinical disease in horses. in the usa, the ap-variant infects goats and deer, but not mice, whereas the ap-ha variant can infect both ruminants and mice under experimental conditions. taken together, these results suggest that distinct a. phagocytophilum ecotypes with varying host tropisms, circulate in europe and the usa. the genetic diversity of a. phagocytophilum must be explored in order to investigate its phylogeny, and to also identify genetic markers capable of distinguishing ecotypes. for this purpose, genome sequences from various animal strains are needed. additionally, given the significant economic consequences of tbf infection in europe, particularly in france, it is important to specifically focus on bovine a. phagocytophilum. within this context, our objective was to sequence and characterize the genome of a. phagocytophilum obtained from a cow with tbf, and generate comparisons with the other available a. phagocytophilum genomes. therefore we followed a whole genome solid-phase sequence capture approach, which allowed us to sequence the genome of a cow sample without the need for strain isolation. this strategy has already been successfully used to sequence the genome of the arthropod symbiotic bacterium wolbachia, another obligate intracellular microorganism. to the best of our knowledge, our study is the first to provide the genome sequence of a bovine strain. following comparison with nine available genomes, we then identified core and accessory a. phagocytophilum genes. core genes comprised the set of orthologous genes shared among the ten sequenced a. phagocytophilum genomes, whereas accessory genes were those orthologous genes not shared among the ten genomes. four genes were specific to the a. phagocytophilum bovine genome, and nine were common to both genomes from domestic ruminants. as all of these genes code for “proteins of unknown function without similarity to other proteins” their functions must now be explored.  confirmation of a. phagocytophiluminfection in february one cow was diagnosed with tbf at the national veterinary school of toulouse. a. phagocytophilum infection was confirmed by the observation of morulas in blood smears and by msp pcr amplification. another cow sample, was confirmed as uninfected by a. phagocytophilum, and was used as a negative control.figure morulas observed in the neutrophils of the bovine blood sample 10_ whole genome capture and sequencing of a. phagocytophilum the whole genome capture step was performed using a nimblegen solid-phase capture array which encompassed,085 bp, corresponding to % of the hz reference strain genome. the library thus obtained was sequenced using the illumina hiseq platform with a paired-end protocol, and a total of, reads were retrieved. subsequently, these reads were aligned to a. phagocytophilum and cattle genomes. a total of, properly paired reads mapped to the pathogen genome, corresponding to % of the total number of reads. this percentage is similar to that obtained by other groups, such as by bright et al.. on the other hand,, properly paired reads were unmapped. after the alignment step, the reads mapping to the cattle genome were discarded and not considered for subsequent analysis. for the subset of reads which mapped to the pathogen genome, further pcr duplicate detection was performed. pcr-duplicated reads are known to arise during the final amplification steps of library preparation, and are related to the reduced complexity of captured libraries. in our case,, duplicated reads were found in the dataset mapping to a. phagocytophilum, which corresponded to a percentage of %. this value is relatively high as compared to those obtained by other authors in similar contexts. for instance, bright et al. reported to have obtained percentages of duplicated reads ranging from % to % in plasmodium vivax field samples, with an average of %. the properly paired a. phagocytophilum reads retained after the removal of duplicates were combined with the properly paired unmapped reads to obtain a final dataset of, reads, roughly corresponding to gb of data and representing a sequencing depth of x. due to the massive amount of raw data, the reads were first digitally normalized using khmer. subsequently,, properly paired reads were retained and then de novo assembled using velvet. the assembly resulted in scaffolds with lengths of 1 kb or more. the n value was  bp with a maximum length of  kb and a minimum length of  bp. the total length of the assembly was, nucleotides, consistent with the sizes of other a. phagocytophilum genomes. after the assembly of the bov-10_ genome, % of the sequences included in the scaffolds aligned to the hz genome. the % remaining sequences within the scaffolds did not map to the genome. these regions could either be absent from the hz genome and/or too variable to correctly align with the hz genome. on the other hand, only % of the hz genome sequence aligned to bov-10_ scaffolds. for the remaining hz regions it was not possible to determine if they were indeed absent from the bov-10_ genome, or if they were missing due to biases introduced during the amplification steps or because of biases related to the capture steps. using bov 10_ new capture probes could be designed, which should improve the capture rate of a. phagocytophilum dna in bovine field samples.figure dot plot of assembled scaffolds versus hz genome. dot plot of contigs versus hz genome. in order to calculate bacterial dna fold-enrichment, we estimated the amount of both a. phagocytophilum dna mass and bovine dna mass in sample 10_ using six different qpcr amplifications: three which targeted a. phagocytophilum genes and three which targeted bovine genes. the relative abundance of the a. phagocytophilum dna within the original 10_ sample corresponded to approximately % of the total dna by mass. since the relative abundance was approximately 59% after the whole genome capture step, the level of enrichment was 197-fold, a rather high value. in fact, results obtained from other similar technical contexts appear to range from ~20-fold to ~100-fold, and in some cases the level of variability among samples was significant. the value obtained in this study could be explained by considering that in our protocol we used 300 μg of cot- bovine dna during the hybridization step, instead of 100 μg as suggested by the nimblegen standard method. this modification could have increased the level of capture specificity by reducing non-specific hybridization.table genes targeted and primers used for quantification of bovine and a. phagocytophilum dna a. phagocytophilum anka glta groel bos taurus gapdh ywhaz ppia a. phagocytophilum dna within the original 10_ sample anka groel glta as previously stated, we obtained approximately 986x genome coverage, an extremely high value, much higher than that required to achieve a whole genome assembly of high quality. such extensive coverage was most likely generated during the experimental planning stage, as we were unable to predict the enrichment level achievable with an initial starting concentration of only % a. phagocytophilum dna. therefore, to ensure that the read number would be sufficient, we used an entire hiseq lane for the sequencing. this first experience shows that it should be relatively simple to sequence several a. phagocytophilum genomes via multiplexing, without compromising the final results in terms of coverage. the only possible issue is that the enrichment ratio could in fact be rather variable from sample to sample, as observed by bright et al.. in conclusion, we have successfully demonstrated the effectiveness of a whole genome capture approach to selectively enrich for pathogenic bacterial dna that was originally mixed with host mammalian dna. this is the first report where an a. phagocytophilum genome sequence has been obtained without any culturing steps. as previously stated, whole genome capture has already been used to sequence the genome of the arthropod non-pathogenic symbiont wolbachia, another obligate intracellular bacterium. here, we demonstrate that whole genome capture is also a suitable approach for pathogenic bacteria wgs such as a. phagocytophilum, and that it could be adapted for large-scale studies of these bacterial genomes, directly from field samples. features of a. phagocytophilumgenomes a. phagocytophilum has a single circular chromosome, without any identified plasmids. the main characteristics of the ten genomes investigated in this study are summarized in table  for bov-10_ the overall gc content was % as for other a. phagocytophilum genomes. this value is very high compared to other rickettsiales-order bacteria, as the genome decay of these bacteria is often accompanied by a low gc% content. a total of cds were predicted, of which were complete. in addition, rrna and trna coding sequences were also identified. overall, genome size appeared to be conserved among the ten strains, whereas the number of genes varied greatly. these differences could be explained, at least in part, by fragmentation of bov 10_ incomplete sequence assemblies. over 70% of the cds had an assigned function.table characteristics of the ten a. phagocytophilum genomes investigated in this study *genes include complete cds, rrna, trna and pseudogenes. phylogenetic analysis phylogenetic trees based on entire sequences of groel, glta, msp and 16s rrna loci were constructed. the two european ruminant strains clustered together in all four phylogenetic trees. the crt and crt ixodes scapularis strains were also associated within the four phylogenic trees, whereas the three human strains, the jm rodent strain and the dog dog strain were always grouped together in the same separate cluster. finally, the mrk horse strain clustered with the human strains only in the cases of 16s rrna and msp phylogenic trees.figure phylogenetic trees based on the groel, glta, 16s rrna and msp4 loci. analyses were performed by the neighbor-joining method using bootstraps. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. comparison of the gene content of other a. phagocytophilumgenomes in order to compare gene content of the ten genomes investigated in this study, we first identified their core and accessory genomes by defining ortholog clusters at the protein level. following analysis, clusters were obtained, of which belonged to the core genome. bov-10_ shared from between to proteins with the other nine strains considered in this study. as for other anaplasmataceae, in which the vast majority of genes belonged to the core genome, we were surprised to observe that a. phagocytophilum contained an exceedingly small proportion of such core genes. this could be due, at least in part, to the application of various annotation methods used for the ten studied genomes, leading to differing gene predictions. in addition, it should be mentioned that only the hz, hz and jm published genomes are fully sequenced, and the genome annotation of the hz strain is of higher quality in comparison to the others, because proteomic data were also used. moreover, clusters were found only in one strain. a large number of these clusters corresponded to the msp2/p gene family. this is not surprising, as these genes tend to have hypervariable central regions, therefore such low global similarity levels prevent their assembly into one cluster. altogether, these factors could have led to a significant underestimation of genes belonging to the core genome. however, nine gene clusters were only detected in the genomes of two european domestic ruminant strains, and four additional clusters were unique to the genome from our bovine sample, when compared to available american a. phagocytophilum genomes. as all corresponding proteins were annotated as "hypothetical proteins", their functions have yet to be explored. for instance, the proteins corresponding to the aforementioned gene clusters could be involved in host tropism for those strains that infect domestic ruminants and/or could be specific to the european strains. in order to examine whether the differences between the european strains are related to animal species or to geographical location, the presence of these genes in a. phagocytophilum genomes from different wild and domestic animals living in the same areas should be investigated. the strategy developed in this paper would be an effective method to rapidly sequence these genomes.figure diagram of the number of proteins shared by different a. phagocytophilum strains. number of proteins shared between bov-10_ and the nine other strains considered in this study small peptides genes encoding small peptides are common features of both prokaryote and eukaryote genomes, and are involved in many biological functions. for staphylococcus aureus, one small peptide class is involved in both virulence and modulation of host immune responses. in bov-10_ we detected cds which coded for small peptides. all predicted small peptides had either no known function or were designated as p44/msp pseudogenes. in total, 31% of predicted cds in the hz genome coded for small peptides. of these small peptide coding sequences, 67% are actually produced, confirming that the majority are not only due to false positive gene prediction during genome annotation. however 82% of the expressed small peptides have unknown functions. in order to better understand the biology of a. phagocytophilum, the functions of these small peptides must be explored. adhesion and host cell internalization genes both the adhesion to and internalization of a. phagocytophilum within host cells are mediated by multiple bacterial adhesins/invasins that cooperatively recognize host cell receptors. to date, at least six a. phagocytophilum adhesins/invasins have been identified. these six proteins belong to the core genome of the ten strains assessed in this study. ompa binds to α -sialic acid of the sialyl lewis x-tetrasaccharide that caps p-selectin glycoprotein ligand-, and msp appears to recognize α- -fucose and/or psgl-1 n-terminal peptide. asp and aipa both recognize an unknown receptor. a. phagocytophilum adhesion could also involve asp and asp two other a. phagocytophilum surface proteins, as indicated by neutralization studies. in bov-10_ and crt strain genomes, we detected a 14-amino-acid-deletion in the n-terminal region of asp the asp domain essential for cellular adherence and invasion is located between the to c-terminal amino acids, therefore this deletion is not expected to have any effect on bacterial adhesion. interestingly, we detected one substitution at position which was only observed in ruminant strains. the ompa protein is completely conserved between strains, except for the domestic ruminant variants; norway variant and 10_ in whom five substitutions were detected. however, as these substitutions lie outside the essential binding domain of ompa, they are also not expected to have any effect on bacterial adhesion. in the hz strain, aipa is a amino acid protein, however in crt dog jm, mrk and hz strains, we detected a deletion of amino acids in the aipa n-terminal region. in strains 10_ crt and hge we detected an additional deletion of four amino acids in the same n-terminal region. however a nucleotide blast search of the hz aipa nucleic sequence revealed that this gene is actually intact in the ten studied genomes. the start codon for this gene is “ttg, therefore this inaccuracy could be due to difficulties with start codon detection during genome annotation. we also detected deletions and substitutions in aipa proteins across the ten strains. further analyses are required in order to draw conclusions on the functional impact of these variations. the hz genome contains one msp locus, two msp homolog loci, and p loci. in order to evade the host immune system, a. phagocytophilum utilizes gene conversion to shuffle msp pseudogenes into the single msp gene expression cassette. one proteomic study has shown that in addition to the expression site aph_ full-length p genes can be expressed at their own loci, whereas silent p genes have to be recombined into the expression site for protein production. the locus expressed during the interaction of msp with psgl- is/are not known. thus it was not possible to further investigate the role of msp2/p variations in a. phagocytophilum binding. moreover the binding sequence on msp remains undefined. asp and asp are the least conserved proteins across all the strains, containing 58/ and 99/ amino acid substitutions or deletions, respectively. however protein characterization studies must still be performed to determine the functional impact of these variations. genes encoding proteins involved in secretion systems protein export systems are extremely important for host-pathogen interactions. gram-negative bacteria often contain the general secretion and the twin-arginine translocation pathways, both involved in exporting protein into the periplasm; and specialized export systems, such as the type iv secretion system, dedicated to exporting specific subsets of proteins. all ten strains studied in this work contained both the sec and tat pathways, as well as type i and type iv specialized export systems. sec pathway all strains contained eight genes of the sec pathway. detection of potential sec substrates was performed as described in the material and methods section, and are listed in additional file many proteins detected as potential sec substrates are surface outer membrane proteins. these proteins may be secreted into the bacterial periplasm by the sec pathway, and then anchored in the outer membrane. the virb virb and virb subunits of the type iv secretion system have also been detected as potential sec substrates. virb and virb proteins are periplasmic and/or outer membrane channel subunits of the t4ss, and virb could be surface exposed in a. phagocytophilum. the sec pathway may secrete these proteins into the bacterial periplasm for assembling, whereas other vir proteins are localized to the inner membrane of a. phagocytophilum, and do not require secretion. seven proteins involved in various cellular processes such as cell metabolism, and two lipoproteins with unknown localization, were also detected as potential sec substrates. ten hypothetical proteins of unknown function were also identified. these proteins could be uncharacterized outer membrane proteins, or might perform their biological activity in periplasmic compartments; consequently their functions need to be explored. the majority of the proteins considered as potential sec substrates were detected in all ten genomes. however some of these proteins were only detected in some of the compared genomes, such as crt38_ which was only present in the crt and crt genomes. tat pathway the tat pathway is found in most bacteria and has been proven to be essential for virulence in several pathogens. in escherichia coli and anaplasma marginale, the minimal set of genes required for tat translocation and a functioning tat system consists of tata, tatb, and tatc. all strains examined in this study contained tata, tatb, and tatc, suggesting that they produce functional tat pathways. it is interesting to note that while tat proteins are not synthesized during hl- cell infection, tata is expressed in tick salivary glands, suggesting that a. phagocytophilum may utilize the tat pathway during tick infection. contrary to many other α-proteobacteria, but consistent across rickettsiae, the three tat genes are dispersed throughout the genome. our in silico investigations led to the prediction of two potential tat substrates across the ten a. phagocytophilum strains. the first had 33% and 28% amino acid identity with the omla protein of azospirillum sp and wolbachia wno, respectively. omla is an outer membrane lipoprotein involved in the maintenance of cell envelop integrity in pseudomonas aeruginosa, and possibly virulence in xanthomonas axonopodis. as lipoproteins are particularly immunogenic and often have adjuvant activity, omla could be a useful vaccine candidate for pathogenic bacteria. as such, it has already been used in pigs to protect against actinobacillus pleuropneumoniae. the fact that omla is present in all the a. phagocytophilum strains studied here, reinforces its potential efficacy as a vaccine candidate against this bacterium. however, this protein is not conserved across all strains. for example, a deletion of approximately amino acids, containing a cysteine residue involved in lipid attachment, is observed in the omla n-terminal region of the hz strain. this observation suggests that omla may not be a surface-exposed protein in the hz strain, raising doubts about the utility of this protein as a vaccine candidate. the second potential tat substrate is a ubiquinol-cytochrome c reductase iron-sulfur subunit involved in the electron transport chain. to date, both the tat and sec pathways have been poorly studied in a. phagocytophilum. due to their importance in other pathogenic bacteria, further investigations are needed in order to fully explore their specific roles in a. phagocytophilum virulence. t1ss secretion system the type i secretion system spans the periplasm and both inner and outer membranes of gram-negative bacteria, and facilitates protein secretion across these compartments. the t1ss is composed of three major proteins: atp-binding cassette transporters, outer membrane factors, and membrane fusion proteins. all ten strains in this study contained these three protein-encoding genes. it remains to be elucidated whether these genes code for an efficient t1ss. however, the strong conservation of the genes encoding this secretion system that has evolved through reductive evolution, observed across all ten a. phagocytophilum genomes,, suggests its importance in the bacterial lifecycle. many t1ss substrates are involved in bacterial virulence. in ehrlichia chaffeensis, three secreted proteins containing tandem repeats and the ankyrin repeat protein, ank involved in host pathogen interactions, are t1ss substrates. to date, no t1ss substrate has been described in anaplasma spp. identifying tss substrates may be a significant step towards a better understanding of a. phagocytophilum biology. t4ss secretion system the type iv secretion system is a multi-protein complex that also spans the periplasm and both membranes of gram-negative bacteria. it can also span eukaryotic host cell membranes and has the ability to transport both nucleic acids and proteins into eukaryotic host cells, and to interfere with host signaling. the t4ss is essential for the survival and virulence of many intracellular bacteria, and the structure of the a. phagocytophilum t4ss has been well studied. each strain studied here contained one copy of virb virb virb and vird4; two copies of virb; virb and virb9; four copies of virb; and a variable number of virb homologue genes. virb proteins constitute the secretion channel and are the most diverse of the t4ss subunits in a. phagocytophilum, both in terms of copy number and sequence. we found homologue virb genes in the hge genome, in hz, hz and 10_ genomes, in dog jm, mrk, and norway variant genomes, in the crt genome, and in the crt genomes. as already described in previous studies, we found that all strains analyzed here lacked both virb and virb genes. the role of t4ss in a. phagocytophilum virulence has been well studied. two a. phagocytophilum t4ss substrates are currently recognized: i) the ankyrin repeat domain-containing protein a, which interacts with host proteins and dna, ii) the anaplasma translocated substrate, involved in the induction of autophagosome formation and potential interference with apoptosis induction. the ats- gene was detected in all ten genomes in this study, whereas anka was absent from the crt genome. as the crt genome was composed of two contigs, separated by a gap, we cannot conclude if anka was really absent from this genome, or if the gene was not sequenced or assembled because it happened to fall within the gap. other secretion systems genes representing components of other secretion systems were not detected in any of the genomes analyzed here, as previously described for the hz genome. however, some proteins, such as aph_ which lack a secretion signal, have been detected on the bacterial surface, indicating that a. phagocytophilum may have alternative motifs directing proteins to cell surface.  in this study we have used an innovative approach in order to sequence the genome of a bovine sample of a. phagocytophilum, and compare it to other published genomes. whole genome capture has already been successfully used for wolbachia genome studies. here we demonstrated that this approach is also applicable to a pathogenic bacterium, a. phagocytophilum, and permits the sequencing of whole genomes without any need for strain isolation. for this reason, we strongly believe that our approach generates a promising tool for large-scale studies of a. phagocytophilum genomes, directly from field samples. genome comparison allowed us to identify four proteins specific to the a. phagocytophilum bovine genome, and nine proteins specific to the two available european domestic ruminant strains. as these proteins could be involved in ruminant strain host tropism, their functions necessitate further exploration. 
our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with a. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains. the four proteins unique to the a. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored. as a. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain. in total, % of obtained reads corresponded to the a. phagocytophilum genome, covering % of the hz genome. dna was extracted from a bovine blood sample infected by a. phagocytophilum. following a whole genome capture approach, a. phagocytophilum dna was enriched 197-fold in the sample and then sequenced using illumina technology. then by performing comparisons with nine previously-sequenced a. phagocytophilum genomes, we determined the core genome of these ten strains. following analysis, coding dna sequences, including complete sequences, were detected in the a. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. using a whole genome capture approach, we have sequenced the first a. phagocytophilum genome isolated from a cow. in domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in europe. anaplasma phagocytophilumcattlecomparative genomicsgranulocytic anaplasmosistick-borne fevertargeted sequence capturewhole genome sequencingissue-copyright-statement© the author 2014 these four coding dna sequences coded for "hypothetical proteins of unknown function” and require further analysis. we also identified nine proteins common to both european domestic ruminants tested. anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks.
 anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks. in domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in europe. as a. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain. our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with a. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains.  dna was extracted from a bovine blood sample infected by a. phagocytophilum. following a whole genome capture approach, a. phagocytophilum dna was enriched 197-fold in the sample and then sequenced using illumina technology. in total, % of obtained reads corresponded to the a. phagocytophilum genome, covering % of the hz genome. then by performing comparisons with nine previously-sequenced a. phagocytophilum genomes, we determined the core genome of these ten strains. following analysis, coding dna sequences, including complete sequences, were detected in the a. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. these four coding dna sequences coded for "hypothetical proteins of unknown function” and require further analysis. we also identified nine proteins common to both european domestic ruminants tested.  using a whole genome capture approach, we have sequenced the first a. phagocytophilum genome isolated from a cow. to the best of our knowledge, this is the first time that this method has been used to selectively enrich pathogenic bacterial dna from samples also containing host dna. the four proteins unique to the a. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords anaplasma phagocytophilumcattlecomparative genomicsgranulocytic anaplasmosistick-borne fevertargeted sequence capturewhole genome sequencingissue-copyright-statement© the author 2014 