The Anaplasma ovis genome reveals a high proportion of pseudogenes

The genus Anaplasma is made up of organisms characterized by small genomes that are undergoing reductive evolution. Anaplasma ovis, one of the seven recognized species in this genus, is an understudied pathogen of sheep and other ruminants. This tick-borne agent is thought to induce only mild clinical disease; however, small deficits may add to larger economic impacts due to the wide geographic distribution of this pathogen. In this report we present the first complete genome sequence for A. ovis and compare the genome features with other closely related species. The 1,214,674 bp A. ovis genome encodes 933 protein coding sequences, the split operon arrangement for ribosomal RNA genes, and more pseudogenes than previously recognized for other Anaplasma species. The metabolic potential is similar to other Anaplasma species. Anaplasma ovis has a small repertoire of surface proteins and transporters. Several novel genes are identified. Analyses of these important features and significant gene families/genes with potential to be vaccine candidates are presented in a comparative context. The availability of this genome will significantly facilitate research for this pathogen.


Background
Anaplasma ovis is a Gram-negative, tick transmitted rickettsial pathogen that causes anaplasmosis of sheep, goats and wild ruminants throughout Asia, Africa, Europe and the U. S. [1,2]. It is typically more pathogenic in goats, and only rarely infects cattle [3][4][5][6][7]. A. ovis infects the erythrocyte where it is phenotypically similar to, but does not provide protection against Anaplasma marginale infection [5]. A. ovis is thought to only induce mild clinical disease, and thus, serious consideration of this pathogen has not been undertaken despite widespread infection in livestock [8]. Losses in productivity, though minor in the individual animal, can be compounded by the fact that infection is pervasive, and the disease state can be exacerbated by stressors such as: co-infections, heavy tick burden, elevated temperatures, vaccination, deworming, and animal movement [2,8]. Therefore, the economic impact of this neglected pathogen may be underestimated.
Anaplasma ovis infection has been detected by examination of Giemsa stained blood smears, complementenzyme linked immuno sorbent assay (cELISA) (Msp 5) and PCR (msp4) [8]. The former two tests, while cheap and simple, do not discriminate to species level, and often a diagnosis of A. ovis as the causative agent is assumed when screening ovine/caprine animals [9]. While the species-specific msp4 PCR has been used in several studies, access to a greater array of diagnostic targets would be of benefit for epidemiological studies and researchers.
Organisms in the order Rickettsiales are small, obligate intracellular bacteria that typically have genomes from 1.2-1.5 Mb [10]. These small genomes are thought to result from reductive evolution and long intracellular association with a host [11][12][13]. The obligate intracellular nature of rickettsial organisms makes them difficult to culture, and to obtain host-free pathogen DNA. The genome of a number of rickettsial pathogens, including representatives of several Anaplasma species, have had their genomes sequenced which has catalyzed research for these pathogens [14][15][16][17]. In this study we have generated the complete genome sequence for the A. ovis Haibei strain using a combination of Solexa and Pacific Biosciences sequencing technologies. The genome has been annotated and manually curated and compared to A. marginale and A. centrale, it's closest relatives [14,16]. The genome features and information about important gene families is presented. We used structural prediction tools to infer novel insights about several hypothetical proteins.

General Features of the A. ovis Genome
The manually curated final genome of A. ovis strain Haibei contains a circular chromosome of 1,214,674 bases and no plasmids, and has an overall G + C content of 49% (Table 1; Fig. 1). Within the seven recognized species in the genus Anaplasma, A. marginale (the type species), A. centrale and A. ovis are more closely related to each other than to the other species in the genus (Fig. 2). A G + C content of 50% is typical for this cluster of related species, with A. phagocytophilum having a slightly lower G + C content. As with all Anaplasma (Rickettsiales) species reported to date, A. ovis contains a single split operon encoding the ribosomal RNA genes, with 23S and 5S genes being transcribed as one polycistron and the 16S gene being transcribed separately. The 37 tRNA genes represent all 20 amino acids (aa). There is a high coding density of 82% and a relatively large average gene length at just over 1 kb.

Pseudogenes
Rickettsial organisms have been described as undergoing reductive evolution, a process whereby they are losing genes over time. The R. conorii genome was described as actively undergoing this process with 37 genes present as "split Open Reading Frames (ORFs)", a process the authors described as the first step in the reductive evolution pathway [12]. The idea being that a gene is mutated with a single base deletion or insertion that results in a frameshift that alters the coding capacity of the gene, (ie, most likely renders the gene non-functional) and a results in smaller open reading frames, in two different frames. Over time, this non-functional gene would acquire more mutations and eventually it would be removed from the genome or changed beyond recognition. The A. marginale St. Maries strain genome contains four such split ORFs, and the Florida strain genome also has four split ORFs, albeit different genes [14,18]. We ruled out sequencing errors as a contribution to the observed changes. Since these A. marginale genomes were completed, high throughput sequencing technologies were developed, which have a tendency to err when sequencing homopolymeric tracks, and these types of frameshift errors are more frequent [19]. When the initial auto-annotation of the A. ovis genome was completed, 47 genes were found to contain frameshifts. Some of these were deemed to be incorrect (the gene was simply annotated in the wrong frame), but all that appeared real were checked by PCR, and corrected, based on the resulting sequence. The final annotation contains seven genes that contain frameshifts (i. e. split ORFs; Table 2). Interestingly, one of the A. ovis split ORFs occurs in mutL (AOV_01085), which also was , and AOV_03945. In the case of genes encoding hypothetical proteins, these frameshifts are recognized as these sequences contain a frameshift as compared to A. marginale; however, it should be noted that when there are not many sequences in the database, these cases should be examined carefully. For example, in the case of AOV_00810, there is a potential ORF in a single reading frame that may be the "real" gene. Other pseudogenes include 20 genes with potential defects to render them nonfunctional. Some are truncated versions of full length genes and others contain internal stop codons (Table 2). Notable among these is a pseudogene for msp1a (AOV_01820) which is truncated at the 5′ end and at the 3′ end. This is the first time we have observed a pseudogene for msp1a in any species. The pseudogene is 2.8 kb upstream from the full length msp1a expression site, with the gene for elongation factor 4 between the pseudogene and the functional msp1a gene. Amongst the other pseudogenes of this type, there was a truncated version of omp14 (AOV_00115); a gene annotated as omp6 (AOV_04995) which is a truncated version of omp10 (AOV_04980), but we retained the name since it is annotated like this in the A. marginale genome; two truncated pseudogenes ([AOV_01970, AOV_01975], [AOV_02415, AOV_02440], [AOV_01020, AOV_01025] [AOV_02120 and AOV_02180] and [AOV_02195 and AOV_02200]) each for infB (AOV 02590), pepA (AOV_03525), thiE (AOV_01015) purA (AOV_03080) and trxB2 (AOV_02200). There were truncated pseudogenes for methylmalonyl-CoA carboxyltransferase (AOV_ 02035), sucD (AOV_00515), GTPase ObgE (AOV_03085), and dihydrolipoyl dehydrogenase (AOV_02835). There was a pseudogene (AOV_05010) for a hypothetical protein that had internal stop codons as compared to the A. marginale homolog. There were two pseudogenes for the virB2 genes, one contained internal stop codons (AOV_04305), while the other was a little shorter at the 5′ end than most virB2s. However, there are alternative start codons that could be used (AOV_04495), i. e. it is possible that this gene could encode a functional product by using an alternative start codon.
Omp13 was auto-annotated as a pseudogene as it contains a stop codon at base 69 relative to the putative start codon. We verified this sequence by amplifying and resequencing this gene. However, it should be noted that there are two potential start codons 84 and 156 bases downstream from the annotated start codon, both downstream from the "internal" stop, and either of these could be the actual true start for this protein. The A. centrale omp13 sequence is shorter than the A. marginale and A. ovis sequences, and alignment of the deduced amino acid sequences for these proteins is shown in Fig. 3. We predict that the conserved Methionine highlighted in Fig. 3 is the appropriate start codon for this protein in all three species. Therefore we have not annotated this as a pseudogene. phagocytophilum: CP000235. Rickettsia felis (CP000053) was used as an out group. The evolutionary history was inferred using the Neighbor-Joining method [61]. The optimal tree with the sum of branch length = 0.23834825 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [62]. The evolutionary distances were computed using the Kimura 2-parameter method [63] and are in the units of the number of base substitutions per site. Evolutionary analyses were conducted in MEGA7 [64]. This tree is representative of the Maximum Likelihood tree A second type of pseudogene was referred to as a "functional pseudogene" during the annotation of the A. marginale genome [14]. The functional pseudogenes are truncated versions of either msp2 or msp3 which, in their current location, cannot express protein, but they can be recombined by gene conversion into the respective expression site to create a new variant of each gene. The St. Maries strain of A. marginale has seven functional pseudogenes for msp2 and another seven for msp3. The number of functional pseudogenes appears to vary somewhat by strain. Anaplasma ovis has seven functional pseudogenes for msp2 and eight for msp3. In addition, there are two classical pseudogenes for msp3, as these genes are so truncated that they do not contain the necessary components for recombination into the expression site (Table 2).
Altogether there are 44 pseudogenes in the A. ovis genome, including genes that contain frameshifts, truncated genes, genes with internal stop codons and the functional pseudogenes for msp2 and 3. We suspect that there are additional pseudogenes in a large family of genes containing a motif (see 'family with motif ' section) as some of the genes are quite truncated and do not appear to be full length compared to the rest of the members of the family. However, as these are all hypothetical proteins, and we do not know their function, we cannot assess whether these truncated gene copies are likely to be functional. Still, a significant portion of the genome, over 3% of the coding capacity (> 40 kb) corresponds to pseudogenes.

Metabolic potential
Anaplasma ovis has the coding capacity for gluconeogenesis but not glycolysis. The genome encodes all the necessary enzymes for the TCA cycle, fatty acid biosynthesis, de novo biosynthesis of purines and pyrimidines, and the nonoxidative pentose phosphate pathway. Many amino acid biosynthetic pathways were not complete. The metabolic reconstruction is very similar to both A. marginale and A. centrale.

Transporters
The genome annotation finds 76 genes/proteins with a role in transport, a similar number to other Anaplasma species (Table 3). The sec pathway for generalized secretion of proteins is present with the exception of secM, a monitoring protein. The twin arginine targeting (Tat) system functions to translocate folded proteins and/or cofactor containing proteins across the membrane. Tat systems consist of two or three subunits integrated into the cytoplasmic membraneeither TatA and TatC or TatA, TatB and TatC [20]. Both tatA (AOV_01285) and tatC (AOV_02605) are present in the A. ovis genome. A gene for TolC is also present in the A. ovis genome. TolC is a multi-purpose pore-forming protein that can be used in the Type I Secretion System (T1SS). TolC is recruited to a membrane fusion protein (MFP) that crosses the inner membrane and bridges it to the outer membrane, after the MFP and an ABC transporter (in the inner membrane) have contacted a substrate [21]. Many T1SSs secrete toxins, and appear to have pairs of MFPs and ABC transporters; however,  centrale sequence, the arrow shows the annotated start codon (GTG), however the sequence upstream from this start is open and contains an in frame methionine start codon. The amino-terminal sequence is not conserved. The methionine at the right most arrow is conserved in all three species, and could be the appropriate start codon for this protein none of these systems have been identified in A. ovis or other closely related species, despite identification of many ABC transporters. Type 4 Secretion Systems (T4SSs) are multimeric protein complexes that span the membrane and secrete proteins (and DNA) into the host cell. The genes encoding the T4SS components are called virB1-11 and virD4, and have also been referred to as rvh to specify rickettsial vir genes. Anaplasmataceae have had an expansion of several genes that make up the complex, such that proteins that normally have a single representative for most bacteria are represented by up to 22 copies in Anaplasma species [22]. Anaplasma ovis has a T4SS that is encoded by 11 virB2 genes, 4 virB6 genes, 2 genes for each of virB4, virB8, and virB9, and one gene each for virB3, virB7, virB10, virB11and virD4. VirB1 and VirB5 have not been identified for any Anaplasmaceae [22]. The virB2 genes come in two types and are annotated as virB2a and virB2b, with the primary difference being that the "a" type are longer at the 5′ end of the gene. The a and b type typically occur in pairs, and usually occur juxtapositioned next to an msp2 or 3 (pseudo)gene. In addition to the 11 functional copies, there are two pseudogenes, discussed earlier. VirB6 is an integral membrane protein and has direct contact with the effector molecules as they are translocated. The expansion of virB6 genes is thought to reflect substrate specificity [23]. Many of the T4SS genes are arranged in two operons, with one containing virB3, virB4, and virB6-1-4, and the other containing virB8-1, virB9-1, virB10, virB11, and virD4. The other genes are dispersed around the genome.

Membrane proteins
Upon using SignalP (version 4.1; [24]) to predict signal peptides, we found that 53 proteins contained signal sequences, and analysis with TMpred [25] indicated that all of these proteins had at least one transmembrane domain. Since this number is much lower than what was predicted for A. marginale (163) using a previous version of the program (version 3), we reanalyzed the A. marginale genome with the current version, SignalP 4.1 and found only 48 sequences predicted to contain signal peptides. This analysis is in line with the relatively small number of proteins that have been predicted to be surface localized for these organisms [14,26].

Gene families of interest
The msp2 superfamily Msp2 and 3 are surface proteins responsible for immune evasion, which they achieve by gene conversion [27,28]. In A. marginale, the msp2/3 functional pseudogenes are recombined by gene conversion into the single expression site to create immune escape variants. The msp2 superfamily is composed of related genes that encode outer membrane proteins (OMPs) that fall into pfam01617. The A. ovis Haibei genome contains 17 members of this superfamily, including omp1,4-8, 10-14, opag1-3, and msp2-4 (not counting functional pseudogenes). Missing from the genome are homologs of omp2, omp3, omp9, and omp15, which are found in A. marginale. Interestingly, the A. ovis complement of omp genes is similar to that of A. centrale. Both species are missing omp2, omp3 and omp15. When it comes to the operon that starts with omp10 and goes through omp6 in A. marginale, the situation is a little different in each species. Recall that Omp7-9 are similar to each other, when comparing across the genes/proteins there are conserved ends and a central variable region: the amino-terminal and carboxy-terminal regions have 85-91% and 81-84% identity while the central regions have 35-51% identity. Anaplasma centrale contains omp10, and a single gene referred to as omp7, followed by a truncated version of omp9. In A. ovis, omp10 is followed by omp8 and omp7, and a truncated version of omp10, which we have called omp6 as in the A. marginale genome. These proteins (omps7-9) are of interest as they have been studied as vaccine candidates [29,30]. As with A. marginale and A. centrale, msp2 is found at the 3′ end of an operon that also contains opag1-3. This operon arrangement is also seen in A. phagocytophilum, but is less conserved, with only two genes upstream of msp2/P44 called omp-1 N and omp-1X (also called p44Sup1) [31]. It is assumed that with a similar number of pseudogenes for msp2 and msp3, that gene conversion will be the operational force working in the A. ovis genome to provide variation in these genes.
In A. marginale, msp3 is also expressed from the 3′ end of an operon, with two virB2 genes at the start of the operon [14]. In the A. ovis Haibei genome, the full length msp3 gene is positioned in close proximity to two virB2 genes; however, the distances between each (160 bp and 290 bp) would suggest these are not typical of a polycistronic arrangement. The deduced amino-terminal sequence has 65% identity with the A. marginale Msp3 sequence.

The msp1 superfamily
Msp1 is a surface protein composed to two polypeptides, Msp1a and Msp1b. The msp1α gene of A. marginale has been used extensively as a surrogate measure for strain diversity [32], and recently, we and others have shown that the corresponding homolog in A. ovis can be used in the same manner [33,34]. Like A. centrale, the Msp1a repeats of A. ovis are longer than those found in A. marginale, with the repeats that have been reported ranging in size from 33 to 47 aa in length. There are four repeats found in the msp1a gene in the A. ovis genome encoding repeats of 49, 55, 55 and 39 aa. While these appear to be longer, we suspect that the full repeat was not reported in the previous papers; when examining the repeats from one of the previous studies, we see that they can also be up to 55 aa in length (see MG642087; from [33]). Similar to the repeats in A. centrale Msp1aS, there are many serine and glutamine residues indicating that the repeats have a polar character. Msp1a is encoded by a single gene; however, as mentioned above, in A. ovis, there is a pseudogene present and care must be taken when analyzing the repeat sequences to ensure that investigators are actually analyzing the repeats from the functional copy of the gene. Downstream from the msp1a gene reside five copies of the mlp gene (mlp1-5) encoding the Msp1a-like protein which have four transmembrane domains that are characteristic of the carboxy-terminus of Msp1a [14].
The A. ovis genome contains one full length copy of msp1b, and a truncated copy, referred to as partial gene 1 or msp1bpg1. The partial gene corresponds to the 3′ 70% of the full length gene. This is a similar arrangement to the A. centrale genome, while A. marginale has two complete genes and three partial genes [35].

The aaap family
In A. marginale, the Anaplasma appendage associated protein (Aaap) associates with actin filaments that are on the cytoplasmic face of the parasitophorous vacuole [36]. This protein is polymorphic among strains, and is characterized by repeats of "EL(K/R/D)AIDA". The St. Maries strain genome sequence revealed two additional aaap-like proteins, or alp1 and alp2, while the Florida strain had a second copy of aaap, and three alp genes [14,18]. The A. centrale genome only had three alp genes, and no gene corresponding to aaap. The A. ovis genome has one aaap and one alp gene. We recently developed an indirect ELISA assay using rAaap for diagnosing A. ovis infection in sheep and goats [37].
Another gene/protein that drew our attention was AOV_02945, which had 14 repeats of 11 aa containing the "ELRAIDA" motif found in Aaap. The protein "hits" with Alp1 of A. marginale with an e score of 1e-23, but all other BLAST hits are below the threshold of significance. AOV_02945 had a conserved domain match to the Neuromodulin_N superfamily [38,39], a family found in multiple malaria adhesins and malaria erythrocyte binding proteins.

AnkA
Ankyrin (Ank) repeats are 33 aa structural motifs that mediate protein-protein interactions, and are more common in eukaryotic proteins that in prokaryotic proteins. It has been observed that Ank repeat containing proteins are often effectors of the T4SS [40]. AnkA (2134 aa), an Ank repeat containing protein, in A. ovis is much longer than the homologs in A. centrale (1424 aa), A. marginale (1387 aa) or A. phagocytophilum (1232 aa). Anaplasma ovis AnkA contains 13 Ank domains, most similarly arranged to the domains found in A. centrale AnkA. The additional~700 aa contains motifs identified in Conserved Domains for both the DnaJ and RNase_E_G superfamilies, which were not found in the shorter copies of AnkA in the other species; however, these hits had relatively low e values (~e-03-e-04). AnkA in A. phagocytophilum is one of the few known effectors of the type IV secretion system, and it translocates to the host cell nucleus where it binds DNA and nuclear proteins. As the other Anaplasma species infect enucleated cells in the mammalian host, it is not clear what role AnkA might play in this setting.

Comparative genomics
Alignment of the A. ovis Haibei genome with A. centrale and A. marginale shows a high degree of synteny between the genomes, although there is a large inversion of~185 kb that spans the putative origin (Fig. 4a). While these genomes are highly conserved, there are a few genes/proteins present in A. ovis that do not appear to have homologs in A. marginale or A. centrale; these are highlighted below.

AOV_01200
The AOV_01200 gene is unique among Anaplasma species genomes. The gene is large, at 4866 bp encoding a protein of 1621 aa. The corresponding protein sequence contains 15 copies of a 46 aa repeat and another 12 copies of a shorter version of the repeat. The deduced aa sequence has highest identity (~37%) to Ice nucleation proteins from Xanthomonas translucens and Pseudomonas syringae. Interestingly, these are both plant pathogens. When we ran structural predictions using I-TASSER, there was a strong match to the RsaA S-layer protein from Caulobacter crescentus [41]. While there was a strong structural match, there was only 13.5% identity at the sequence level. Interestingly, both the ice nucleation proteins and the RsaA S-layer protein form hexamer complexes, and the matches with these proteins may reflect the conserved regions that are integral in the hexamer complex formation rather than the actual function of the protein [41][42][43].

AOV_01195
AOV_01195 encodes a protein of 487 amino acids, with best hits to AM366 in A. marginale or ACIS_00940 in A. centrale, although these genes encode proteins with > 2800 amino acids. The match is~48% identity for a span of 175 amino acids. All of these proteins have no known function.

Family with motif
There is a family of genes/proteins that are found in four clusters throughout the genome that contain a peptide motif corresponding to the sequence "ISAVAAVAY LAVTGVSIRDLYRSCKQVIQVKEEGLVTVQSLQPVLTP ITPIAGKINYGKIASA". This motif typically occurs near the amino terminus, and the longer genes/proteins have low similarity at their carboxy-termini. The members of the family vary tremendously in size, with a few of the members of the family being quite short and which do not contain the motif, but match to other parts of deduced sequences within the family. It appears that some of these genes may be degraded forms of the longer genes; however, since we know nothing about the functionality of these sequences, we have maintained them as CDSs rather than marking them as pseudogenes. The clusters are as follows: 1) AOV_01045 (512 aa) and AOV_01050 (782 aa); 2) AOV_02420 (71 aa), AOV_02425 (118), AOV_02430 (334 aa), AOV_02450 (510 aa), with each of the smaller genes matching to different regions of AOV_02450, although they are not exact matches, and together do not correspond to a full length AOV_02450. Only AOV_02425 and AOV_02450 contain the motif. 3) AOV_02730 (437 aa), AOV_02735 (298 aa), AOV_02740 (356 aa), AOV_02745 (308 aa), AOV_02750 (80 aa), AOV_02755 (472 aa), AOV_02760 (318 aa), with only AOV_02750 missing the motif, but matching the carboxy-terminus of AOV_02755. 4) AOV_03535 (83 aa), AOV_03540 (277 aa), AOV_03545 (341 aa), AOV_03550 (475 aa), with AOV_03535 missing the motif, but matching the carboxy-terminus of AOV_02430. In pairwise comparisons, the proteins range from 0 to 89% identity with the highest match between any two proteins ranging from 62 to 89%. Altogether there are 17 genes/proteins in this family, which contrasts with A. marginale which has just four members of the family, AM673, AM676, AM773, and AM959. Interestingly, A. centrale has 10 members of the family (ACIS_00311, ACIS_00381, ACIS_00561, ACIS_00637, ACIS_00674, ACIS_00675, ACIS_00677, ACIS_00679, ACIS_00684, and ACIS_00977), however; the protein motif is shorter in this species.

The Idaho sequence
Recently, a draft genome sequence (accession number PKOE00000000) was deposited in GenBank reported to be the A. ovis Idaho strain [44]. The history of this strain is that it was isolated from sheep in 1988 by feeding Dermacentor andersoni ticks on naturally infected sheep and transmitting to naïve, splenectomized sheep, thus demonstrating tick transmittability [45]. The strain was isolated prior to the development of molecular tests, and (we believe) there was an assumption that if it came from a sheep, it was A. ovis. The cELISA based on Msp5 will not discriminate between Anaplasma species [45,46]. However, with the development of molecular tests, there is a clear discrimination between A. ovis and A. marginale using genes such as msp4, and in fact, in 2002, when the Idaho isolate was first put into tick cell culture it exhibited an A. ovis Msp4 sequence indistinguishable from other published A. ovis Msp4 sequences [47,48]. The Idaho draft sequence is in 43 contigs, which we BLASTed against A. marginale and our A. ovis Haibei sequences finding 99% and~88% sequence identity, respectively. We bioinformatically "stitched" most of the Idaho contigs together and created a pseudochromosome (small contigs of~1 kb each were left out), which was used for alignment with both an A. marginale genome and the A. ovis Haibei genome (Fig. 4b).
Upon closer inspection, the Idaho genome contains an msp1α gene indistinguishable from A. marginale Virginia strain msp1α, encoding two repeat sequences: AB. We and other researchers have found that the form of the repeats varies between these species (discussed above), with A. ovis having much longer repeats similar to A. centrale. Further, the Msp4 sequence of the Idaho genome has 96-97% aa identity with multiple Msp4 sequences from A. ovis, and 100% identity with Msp4 sequences from A. marginale (data not shown). The 16S rRNA gene from this genome segregates with A. marginale upon phylogenetic analysis (Fig. 2). Finally, in the same paper reporting the Idaho genome an A. marginale strain Oklahoma-2 genome is also reported (accession number PKOF00000000). Strain Oklahoma-2 (Wetumka) has an msp1α genotype of KCH [49], however the genome sequence also contains an msp1α gene that has a repeat structure of AB. The Oklahoma-2 genome is in 44 contigs of surprisingly similar size to the Idaho contigs. For example, the three largest contigs from the Idaho genome are 305,977, 85,373 and 71,461 bp, while for Oklahoma-2 they are 306,104, 85,373 and 69,474 bp. There are 3 bp mismatches between the two contig 1 s from each sequence, 1 mismatch between the contig 2 s and 0 mismatches between the contig 3 s. This level of sequence identity is unprecedented for two sequences of A. marginale that are different strains, let alone two sequences that are different species [18,50]. It appears that the same genome was sequenced twice. Further, with the Msp1a, Msp4 and 16S gene/protein data presented above, the most parsimonious explanation is that the "Idaho" sequence is actually an A. marginale sequence similar to the Virginia strain.

Discussion
The availability of a genome sequence for A. ovis will facilitate the development of better diagnostic tests and a vaccine for this pathogen. When compared to other Anaplasma species, A. ovis displays marked similarities to both A. marginale and A. centrale, with some genes/ regions being more similar to one species and other genes/regions being more similar to the other. An overall theme of conserved metabolic pathways and conserved synteny was obvious, with the exception of the large 185 kb inversion. Both A. ovis and A. centrale cause mild disease in their respective hosts as compared to A. marginale, which can be much more virulent. This difference in pathogenicity is a subtle genomic difference that has yet to be elucidated. There are no genes in A. marginale that are absent in these other two agents that provide overt clues to the differences in virulence. More pseudogenes were detected in the A. ovis genome as compared to A. marginale and A. centrale, but this is not surprising, as when the first A. marginale genome was sequenced there were not many close relatives to compare with, and the comparison of A. ovis to the other sequenced Anaplasma spp. helps to highlight the pseudogenes. However, even with this caveat, there appears to be more fragmented genes in A. ovis than in A. marginale and A. centrale. Despite growth in the databases since the first Anaplasma genome was completed, a large fraction (26%) of genes/proteins were still annotated as "hypotheticals", or proteins of unknown function with no known homologs. The use of tertiary structural mapping was used to identify novel insights for a protein of unknown function that is unique among the Anaplasmataceae. The genome sequence has already been used to develop a novel ELISA and to design msp1a PCR assays for strain differentiation, and we expect that further advances will be enabled by having this genome available.

Conclusions
Anaplasma ovis is an understudied ricketsial pathogen of ruminants that is closely related to A. marginale and A. centrale. This is the first complete A. ovis genome sequence which demonstrates a high degree of synteny with closely related Anaplasma species. Indeed, many features of the genome are conserved with these close relatives, such as genome size (1.2 Mb), the split operon arrangement for ribosomal RNA genes, metabolic potential and small repertoire of surface proteins and transporters. What stands out is the larger number of pseudogenes encoded in this genome as compared to it's close relatives and several novel genes not seen in other Anaplasmataceae.

Origin of Anaplasma ovis Haibei isolate
Anaplasma ovis strain Haibei was first detected and identified by light microscopy examination of thin blood smears from a dying sheep in Haibei County in Qinghai Province. In addition, amplification with MSP45/MSP43 primers was performed according to [51]. Five ml of blood from the infected sheep was collected into a sterile EDTA-K 2 anticoagulant tube and inoculated into a splenectomized sheep via the jugular vein as soon as the blood arrived at the laboratory. Blood from the experimental sheep was examined daily by light microscopy. When the bacteremia reached 15%, blood was collected and 5 ml aliquots, supplemented to 8% dimethyl sulfoxide (DMSO) and cryopreserved in liquid nitrogen.

Propagation of a. ovis
Two three-month-old sheep were purchased from Chengye farming cooperative in Jingtai County, Gansu Province. The sheep were screened for the absence of A. ovis, Babesia and Theileria for a month before conducting animal experiments by weekly examination of blood smear using light microscopy and previously described PCR protocols specific for each pathogen [51][52][53]. Sheep No. 007 was held as a backup in case the necessary samples were not obtained from sheep No. 008. Sheep No. 008 was splenectomized to ensure rapid initiation and propagation of the infection. Infection was by intravenous inoculation of 10 ml of the cryopreserved, A. ovis-infected blood (approximately 15% bacteremia). When the bacteremia reached approximately 15%, venous blood from the sheep was harvested in a sterile flask containing anticoagulant (EDTA). Sheep No. 008 died from infection 3 days after a blood sample was collected for the project.

Bacterial purification
Bacterial purification was as previously described [37]. Red blood cells (RBCs) were separated by centrifugation at 1000×g for 10 min, and the upper layer containing the white blood cells (WBCs) was discarded. The packed RBCs were suspended in phosphate-buffered saline (PBS, pH 7.2), and the remaining WBCs were removed using a commercial leucocyte filter (Nanjing Shuangwei Biotechnology, Nanjing, China). The flow-through was centrifuged as above, and the supernatant was discarded. The harvested RBCs were suspended in four volumes of PBS containing 7% glycerin and placed at room temperature for 30 min, and then centrifuged again to harvest the RBCs. The cells were then added to a flask containing four volumes of physiological saline to let the cells lyse completely. The lysate was centrifuged at 1000×g for 10 min to remove cell debris. The supernatant was then centrifuged at 10,000×g for 30 min to pellet the bacteria. The pellet was washed three times with physiological saline by centrifugation at 10,000×g for 10 min. The resulting pellet was used for DNA preparation.

DNA preparation
DNA was extracted using a genomic DNA extraction kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions resulting in 1 ml of sample in Elution buffer. The DNA concentration was 296 ng/μl with an OD260/280 ratio of 1.92 using a Quant-iTTM dsDNA HS Assay Kit (Thermo Fisher Scientific, Beijing China).

Genome sequencing
A 20-kb genomic DNA library was prepared suitable for P6/C4 chemistry using the SMRT bell template preparation kit 1.0 according to the manufacturer's protocol. The A. ovis Haibei genome was sequenced using the PacBio single-molecule real-time (SMRT) sequencing technology using one SMRT cell on the PacBio RSII sequencing platform (Pacific Biosciences, Menlo Park, CA, USA; BGI-Shenzhen, Shenzhen, China). A total of 76,050 reads with a mean read length of 8372 bp were obtained. To minimalize the single-pass error generated by the PacBio sequencing, three DNA libraries were constructed with the insert sizes of 500 bp, 2000 bp, or 6000 bp, and were sequenced according to the standard protocols for the Illumina Hiseq 4000 platform (BGI-Shenzhen, Shenzhen, China). A hybrid approach was used to assemble and finish the genome using hierarchical genome-assembly process (HGAP) and automated workflows [54].

Annotation
The assembled genome was submitted for annotation to the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [55]. This pipeline uses a series of programs to call genes, predict proteins, assign functional annotation, identify frameshifts, and non-coding RNAs. This annotation was manually curated, several frameshifts that were detected on the first pass were checked and corrected, where necessary. The metabolic potential was assessed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) [56]. The transporters were analyzed using TransportDB 2.0 [57]. Repeats were assessed using Rapid Automatic Detection and Alignment of Repeats (RADAR) in protein sequences [58]. Protein structures were modeled with I-TASSER [59]. Whole genome alignments were done using Artemis Comparison Tool [60]. MEGA7.0 was used to generate phylogenetic trees. Functional pseudogenes are truncated copies of msp2 or msp3 and are annotated from stop codon to stop codon. Classical pseudogenes were detected either due to a frameshift (addition or deletion of a single base pair) within an otherwise complete gene, or when the PGAP annotated a truncated gene as an incomplete version of a known, full length gene.
The genome sequence has been deposited in GenBank with Accession number CP015994.