Genome Degradation in Brucella ovis Corresponds with Narrowing of Its Host Range and Tissue Tropism

Brucella ovis is a veterinary pathogen associated with epididymitis in sheep. Despite its genetic similarity to the zoonotic pathogens B. abortus, B. melitensis and B. suis, B. ovis does not cause zoonotic disease. Genomic analysis of the type strain ATCC25840 revealed a high percentage of pseudogenes and increased numbers of transposable elements compared to the zoonotic Brucella species, suggesting that genome degradation has occurred concomitant with narrowing of the host range of B. ovis. The absence of genomic island 2, encoding functions required for lipopolysaccharide biosynthesis, as well as inactivation of genes encoding urease, nutrient uptake and utilization, and outer membrane proteins may be factors contributing to the avirulence of B. ovis for humans. A 26.5 kb region of B. ovis ATCC25840 Chromosome II was absent from all the sequenced human pathogenic Brucella genomes, but was present in all of 17 B. ovis isolates tested and in three B. ceti isolates, suggesting that this DNA region may be of use for differentiating B. ovis from other Brucella spp. This is the first genomic analysis of a non-zoonotic Brucella species. The results suggest that inactivation of genes involved in nutrient acquisition and utilization, cell envelope structure and urease may have played a role in narrowing of the tissue tropism and host range of B. ovis.


Introduction
Although previous studies have supported the notion of Brucella as a monospecies genus with different biotypes [1], it is still largely accepted that the genus Brucella is divided into six species, named according to their preferential hosts. Each one of the species is hostadapted, but not host-restricted [2][3][4]. Four of the six Brucella species, namely B. melitensis, B. abortus, B. suis, and B. canis, are capable of causing human disease. B. melitensis and B. suis are the most pathogenic, whereas B. abortus is considered of moderate pathogenicity, and B. canis is considered of low pathogenicity for humans. There are no reports of human infections with B. ovis or B. neotomae [3]. In addition to the six classical Brucella species, Brucella has also been isolated from marine mammals, and two species, B. pinnipedialis and B. ceti have been proposed [5]. Brucella isolates from marine mammals can cause human infections, with one reported case of infection due to laboratory exposure [6], and two reported cases of natural infections resulting in neurological disease [7].
Brucella ovis was initially recognized in the beginning of the 1950's in New Zealand and Australia as a bacterial agent associated with epididymitis and abortion in sheep [8]. Since then this organism has been isolated in several countries [9], and is considered one of the most important causes of ovine infertility, with a significant economic impact on the sheep industry [10]. B. ovis has a worldwide distribution in areas where sheep are economically significant, with the exception of the Great Britain [9]. The prevalence in herds ranges from 9.1 to 46.7% [11], and the seroprevalence within positive herds varies between 2.1 to 67% [11][12][13][14].
B. ovis is stably rough, and it is one of the two classical Brucella species that do not have zoonotic potential. In sheep, the organism causes either clinical or sub-clinical chronic infections characterized by epididymitis, orchitis, male infertility, and occasionally abortion in pregnant ewes [15]. Sexually mature rams are more susceptible than young males [16]. However, B. ovis infection may affect males as young as 4 months old [9]. Natural transmission apparently occurs through mucosal membranes, and venereal transmission is significant when a female previously mated with an infected male copulates with a second susceptible male during the same period of estrus [17]. Upon invasion through mucosal membranes, B. ovis initially resides in local lymph nodes, which is followed by bacteremia and finally colonization of the genital tract around 30 days post infection [18]. The factors defining the genital tropism of this organism remain unknown.
Sequencing of B. melitensis, and B. suis genomes demonstrated a high level of similarity between the two genomes, with over 90% of the genes having more than 98-100% nucleotide identity [19]. Furthermore, comparison between these two species resulted in the identification of only 32 and 43 genes that were unique to B. melitensis and B. suis, respectively. This level of variability is remarkably low even when compared to variations between serotypes within the same bacterial species such as in serotypes Typhi and Typhimurium of Samonella enterica [20]. More recently, the complete genome sequence of B. abortus (strains 9-941 and 2308) became available confirming the striking similarity both among different species of Brucella and within the species B. abortus [21,22]. Comparisons between these three Brucella species revealed more than 94% identity at the nucleotide level. In addition, comparisons between the genomes of the two B. abortus strains that have been sequenced (9-941 and 2308) resulted in identification of only a small number of strain-specific deletions and polymorphisms [21]. The genetic similarity among Brucella species has been confirmed by whole genome hybridizations [23]. Together these studies support the original hybridization studies performed more than 20 years ago suggesting that Brucella is a monospecific genus from the genetic point of view [1]. Considering the high level of identity among Brucella species pathogenic to humans, the comparison of those species with a Brucella lacking the potential to cause human infections may bring new insights into host specificity and pathogenesis. In this study we present the genome analysis of the rough non-human-pathogen Brucella ovis and compare its genome with those of the zoonotic Brucella spp. Our analysis focused on two sets of genomic features (i) deletions and gene degradation that could potentially result in loss of virulence factors important for infection of hosts other than sheep, and (ii) B. ovis-specific genes that could contribute to genital tract tropism or cause a reduction in virulence for non-ovine hosts.

Results and Discussion
General features of the B. ovis genome The B. ovis type strain ATCC25840 (also known as 63/290 or NCTC10512) used for sequencing was isolated from a sheep in Australia in 1960 [8]. The genome of this strain consists of two circular chromosomes of 2,111,370 bp (Chromosome I; NCBI Accession Number NC_009505) and 1,164,220 bp (Chromosome II; NCBI Accession Number NC_009504), which are predicted to encode a total of 2890 proteins, 1928 on ChrI and 962 on ChrII ( Table 1). Features of the B. ovis genome are summarized in Table 1. Comparison with the sequenced genomes of B. suis (GenBank Accession numbers NC_004310 and NC_004311) [24], B. abortus (GenBank Accession numbers NC_007618 and NC_007624) [21,22] and B. melitensis 16M (GenBank Accession numbers NC_003317 and NC_003318) [19] shows a large degree of conservation, particularly in the % G+C content and size of the chromosomes. This comparison also revealed several speciesspecific differences, including regions missing from B. ovis relative to the other sequenced Brucella genomes and genes unique to B. ovis. These differences are listed in Table 1 and Figures 1-2, and are discussed below.
Comparison to B. suis, B. abortus, and B. melitensis proteomes There is a significant degree of overlap between the predicted proteomes of sequenced Brucella species. Comparative best-match blastp searches identified 2282 annotated proteins in B. ovis that are shared with B. suis, B. melitensis and B. abortus, 79% of the annotated proteome. Nonetheless, this analysis suggested that the B. ovis genome lacks 675, 610 and 539 protein coding genes annotated in B. suis, B. melitensis and B. abortus 2308, respectively (Table S3). To determine whether these genes are truly absent from the B. ovis genome they were compared to the B. ovis chromosomal sequences using blastn searches. Interestingly, good matches were found for 64, 125 and 18 genes thought to be lacking in B. ovis in the genomes of B. suis, B. melitensis and B. abortus 2308, respectively (Table S3). There are several possible reasons for these genes not being highlighted as shared using comparative best-match blastp searches, e.g., they may be pseudogenes in B. ovis and therefore, not part of the predicted proteome, they may be the products of duplications where only one duplication product is matched in best match searches or differences in gene annotation between the strains resulted in these sequences not being annotated as a protein coding gene in B. ovis. In total only 33 annotated protein coding genes in B. ovis are unique to this species.

Genomic rearrangements
Gradient figures were used to compare the chromosomes of B. ovis with those of the other sequenced Brucella genomes. No large inversions or rearrangements were observed in the B. ovis genome compared to the sequenced genomes of B. suis or B. melitensis ( Figure 1).

Genomic deletions
Chromosome I. Four deletions of 4 kb or larger were identified in the B. ovis genome, compared to its closest relative, B. suis. Chromosome I lacks a 15 kb region that encompasses B.
suis BR0966-BR0987. Interestingly, in B. suis, this region is inserted into the sequence of tRNAgly and is adjacent to a phage integraselike gene, features suggestive of horizontal gene transfer. This region contains the wboA glycosyl transferase gene, shown to be essential for lipopolysaccharide biosynthesis [25], and a second glycosyl transferase, as well as several hypothetical proteins. The lack of the two glycosyl transferases likely contributes to the rough LPS phenotype of B. ovis. These findings were in agreement with previous reports by Vizcaino et al and Rajashekara et al [23,26] showing the absence of these genes from B. ovis.
A second deletion on Chromosome I of 7745 bp encompasses BR1078-BR1083. This island in B. suis contains three hypothetical genes and two site specific recombinases and is flanked by two tRNA-Leu genes, of which one remains in B. ovis. A smaller deletion of 3954 bp on Chromosome I has led to loss of two genes (BR1852-BR1853) encoding a transcriptional regulator and a protein predicted to be a branched chain amino acid permease. This deletion is also associated with transposable elements, as these two genes in B. suis are flanked by copies of IS2020 [27], one of which remains in B. ovis.
Chromosome II. The 44.5 kb island in B. suis (BRA1074-BRA1113; [28]), containing four predicted ABC transport systems and three transcriptional regulators, is absent from the B. ovis genome. The presence of two copies of IS1239 flanking two pseudogenes in B. ovis suggests that this is the result of a loss of this region by recombination. The 18.3 kb IncP island on B. suis Chromosome II containing BRA0362-BRA0379, previously reported to be present in B. suis biovars 1-4, B. canis, B. neotomae, and marine mammal isolates but missing in B. melitensis [24,28], is also absent from the B. ovis genome, as reported by Lavigne and colleagues [28].

Unique genomic regions
Chromosome II, contains an island of 26.5 kb (BOV_A0482-BOV_A0515) with structure suggestive of a composite transposon (Fig. 2). This island likely overlaps the B. ovis-specific 21-kb SpeI fragment of the small chromosome identified previously by genome mapping [29]. Proteins encoded on the island include transposases, an ABC transporter, a putative hemagglutinin, and several hypothetical proteins. This region is present in 17/17 B. ovis strains tested, suggesting a high level of conservation within the species (Supplementary Table S2). Interestingly, the predicted product of BOV_A0497 exhibits similarity to antitoxins of toxinantitoxin maintenance systems [30], suggesting a possible selective pressure for maintenance of this genetic island in B. ovis. Genome sequence data and analysis of the island by PCR showed that this region is absent from the genomes of B. suis, B. abortus, B. melitensis, B. canis and B. neotomae (Table 2), suggesting initially that it may be specific to B. ovis. However, we detected this island in three marine isolates of Brucella from captive bottlenose dolphins [31,32]. The protein coding genes within this unique region constitute the majority of the unique protein coding genes in B. ovis (Supplementary Table S3).

Transposable elements
The genome of B. ovis contains 38 complete copies of IS711, confirming previous estimates obtained by hybridization [33]. 25 of the copies are located in Chromosome I, and 13 in Chromosome II. Interestingly, several of these copies appear to be expanded clonally, suggesting that they could be active in B. ovis. In 17 cases, IS711 is inserted in copies of a repeated sequence of the BruRS family [34]. Five of the IS711 copies appear to disrupt genes, which could be a contributing factor to the general process of genome degradation observed in B. ovis. A case of special interest is the B. ovis-specific island (see above), where a 25 kb region between two copies of IS711 in B. ovis is absent in all the other sequenced species, suggestive of either deletion by recombination between the two copies in the other Brucella species, or of acquisition of the region with duplication of IS711.

Pseudogenes
It has been proposed that the unique complement of pseudogenes in each of the Brucella species may contribute to their differing degrees of infectivity and host preference [21,22]. Interestingly B. abortus, which is less virulent for humans than B.  Table 1). Similar to B. abortus, the B. ovis genome contains a large number of pseudogenes with 244 identified. The small chromosome contains more pseudogenes (125; 11.3%) than the large chromosome (119; 5.7%) ( Table 1). Of the 244 B. ovis pseudogenes, 40 are hypothetical or conserved hypothetical genes, 62 have predicted transport functions, 23 are defective transposases, and 14 are predicted to have regulatory functions. The finding that some pseudogenes in the B. melitensis flagellar region encode full-length proteins raises the question of whether some pseudogenes in B. ovis may also be functional [35].

Metabolism
Urease. Based on biotyping, B. ovis is urease-negative. However it contains the two urease clusters described in all the other sequenced Brucella genomes. Urease has been reported to be important for the ability of B. abortus [36] and B. suis [37] to survive passage through the stomach in the mouse model of infection. The importance of urease for oral infection is consistent with a lack of human infections observed with B. ovis, despite identification of this organism in the milk of infected ewes [38]. The ure1 cluster is the only one showing urease activity at least in B. abortus, B. melitensis, and B. suis [36,37], while the ure2 does not have any demonstrable urease activity. The ure1 cluster of B. ovis contains several point mutations that are predicted to result in conserved changes shared by at least one other urease positive species. However, UreC1 contains a 30 bp deletion that would cause a loss of 10 amino acids in UreC1. Although all the residues described to be important for enzymatic activity of UreC1 are conserved [39], this deletion must render the urease inactive. Moreover, complementation with the ureC1 gene from B. melitensis (Sangari, unpublished results) results in urease activity. Regarding the ure2 cluster, ureF2 (BOV_1316) and ureT (BOV_1319) are pseudogenes, while the remaining genes are conserved, suggesting that this cluster could encode an unknown activity.
Sugar metabolism. B. ovis is defective in oxidative metabolism of arabinose, galactose, ribose, xylose, glucose and erythritol [40]. Analysis of the genome sequence reveals a possible basis for these metabolic defects in B. ovis relative to the human pathogenic Brucella species. Several putative sugar transporters predicted to be functional in other Brucella species appear to be inactivated by frameshifts, point mutations or gene degradation in B. ovis. Further, pckA encoding phosphoenolpyruvate carboxykinase (BOV_2009) is inactivated by frameshift, which would affect the gluconeogenesis pathway and the ability of B. ovis to utilize pyruvate, amino acids, or glycerol as carbon sources.
Erythritol. Erythritol is the preferred carbon source of B. abortus [41,42]. Erythritol is metabolized in Brucella by the enzymes encoded in the eryABCD operon [43] Moreover, it has been recently described that the carbohydrate transport system located upstream of the ery operon constitutes the erythritol transport system in Rhizobium leguminosarum bv. Viciae [44], and that the operon immediately downstream also forms part of the erythritol catabolic pathway. Microarray experiments have revealed that these three operons are regulated by erythritol in B. abortus, reinforcing that the three clusters participate in erythritol catabolism (Sangari et al, unpublished). B. ovis does not oxidize erythritol, and it is not inhibited by its presence in the growth media. This is reflected in the genome by the presence of a stop codon in eryA (BOV_A0811) and a frameshift in eryD (BOV_A0814) that render them pseudogenes. In addition two genes of the putative ABC erythritol transport system, eryF and eryG (BOV_A805 and BOV_A806)) carry mutations that render them pseudogenes (a 2 bp deletion resulting in an premature stop codon). The mutation in eryD could have an effect in the over expression of all genes of the ery system. On the contrary, mutations in the transport genes and in eryA block the entry of erythritol in the cell and its phosphorylation, avoiding the accumulation of toxic intermediates and the depletion of ATP observed in the vaccine strain S19 [45]. The accumulation of mutations in these two clusters suggest that they may no longer be needed by B. ovis. The third cluster is intact, and the enzymes use substrates that are central (or core) carbohydrate metabolites such as dihydroxyacetone phosphate, glyceraldehydes-3-phosphate, and other three carbon compounds that can be produced after decarboxylation of erythritol and its derivatives. While it has been hypothesized that erythritol may serve as a carbon source during growth of B. abortus, B. suis, and B. melitensis in the placenta of their natural hosts, an analysis of deletion mutants in these models has not been reported. However, the lower incidence of abortion in sheep flocks infected with B. ovis compared to B. melitensis correlates with the inability of B. ovis to use erythritol as a carbon source. Mutants in eryB and eryC have been described to have a limited ability to grow in macrophages [46]. This limitation may well contribute to the limited virulence of B. ovis.
Glucose and Galactose. Unlike B. melitensis, B. abortus and B. suis, B. ovis is unable to grow on glucose or galactose as a primary carbon source [40]. Analysis of genes involved in uptake and utilization of these carbon sources reveals that B. ovis gluP (BOV_A0172), encoding a glucose/galactose transporter [47] is a pseudogene. B. abortus gluP mutants have a reduced ability to persist in the spleens of mice [48], suggesting that the ability to utilize these carbon sources may be important for systemic persistence of the human pathogenic Brucella species. However B. suis also lacks GluP and is able to oxidize glucose and galactose, suggesting that additional functions are lacking in B. ovis that contribute to utilization of these carbon sources. Several predicted sugar ABC transporters (BOV_1299-BOV1301, BOV_A0278-BOV_A0282, BOV_A0645-BOV_A0648 and BOV_A1083-BOV_A1086) of unknown specificity contain pseudogenes, which may potentially reduce the ability of B. ovis to take up glucose and galactose.
Ribose. Two components, a permease and an ATP binding protein of a putative ribose ABC transport system (BOV_A0936-BOV_A0937) are pseudogenes in B. ovis, suggesting that the inability of B. ovis to utilize ribose as a sole carbon source may be the result of a reduced capacity to take up ribose from the growth medium. Similarly, a periplasmic binding protein and an ATP binding protein of a predicted ABC transporter for xylose (BOV_A1055-BOVA1056) contain frameshifts, which may underlie the inability of B. ovis to utilize xylose [40].
Oxidase phenotype. B. ovis is the only Brucella species with a negative oxidase phenotype. Oxidase phenotype depends on the activity of cytochrome C oxidase which in B. suis is encoded by at least 7 genes, four of them organized in one operon, BR0363-BR0360 (ccoNOQP), together with BR0467 (coxB), encoding cytochrome c oxidase, subunit II, BR0468 (coxA) encoding cytochrome c oxidase, subunit I and BR0472 (coxC) encoding cytochrome c oxidase, subunit III. In B. ovis, the operon ccoNOQP (BOV_0376-BOV_0379), encoding the cb type cytochrome c oxidase is well conserved, except the gene ccoO (BOV_0378), which contains a frameshift generated by deletion of an A near its 59 end, is a pseudogene. The B. ovis coxB gene (BOV_0473) contains a frameshift that very probably inactivates the gene, while coxA (BOV_0474) differs only in two residues with the B. suis product. The last 6 amino acids of coxC (BOV_0478) are lost as result of a short deletion (56 bp) that fuses this product with the next, apparently unrelated Orf. A short repeated sequence (GGGGCGGC) at both ends of the deletion seems to be responsible for this rearrangement. These genomic differences may be responsible for the oxidase negative phenotype of B. ovis.
Nitrogen metabolism. Several genes encoded in the B. suis genome with inferred functions in nitrogen metabolism are not predicted to be functional in the B. ovis genome. The genes norB (BOV_A0225), encoding the large subunit of nitric oxide reductase, and nosX (BOV_A0256), a gene of unknown function in the operon encoding nitric oxide synthase, are pseudogenes in B. ovis. The third gene, narK (BOV_A0276), encoding a nitrite extrusion protein, is degenerate, as was found in the B. melitensis and B. abortus 2308 genomes [21]. Nitric oxide reductase is required for survival and persistence of B. suis in mice [49], suggesting that lack of this activity in B. ovis may contribute to its restricted tissue tropism and host range.
A locus that contributes to growth of E. coli on aspartate as a nitrogen source, xanthine dehydrogenase (BOV_0365-BOV_0367; [50]) contains a pseudogene in B. ovis, suggesting a further defect in nitrogen metabolism. Further, this locus could function in salvage pathways for purine nucleotides, which could contribute to nitrogen metabolism. A correlation between a defective purine nucleotide salvage pathway and reduced ability of B. ovis to survive intracellularly would be consistent with the importance of purine biosynthetic pathways for full virulence of B. abortus and B. melitensis [51,52]. Further, a homolog of Sinorhizobium meliloti fixI (BOV_0373), encoding an cation pump involved in symbiotic nitrogen fixation [53], is inactivated by a point mutation in the B. ovis genome. As the function of the fixGHI genes in Brucella spp. has not been determined, the biological significance of this gene inactivation for B. ovis is currently unknown.

Host-pathogen interactions
Lipopolysaccharide. B. ovis is known to have a rough LPS phenotype, which given the critical role of O-antigen in pathogenicity of B. abortus, B. suis and B. melitensis, likely contributes to its reduced pathogenicity for laboratory animals compared to other Brucella spp. [8,54,55]. As mentioned above, the wboA gene is missing from B. ovis, as well as a second, genetically linked glycosyltransferase. Pseudogenes with a possible function in LPS biosynthesis include a glycosyltransferase (BOV_A0475), an LpxA family acetyltransferase (BOV_A0367) and a putative undecaprenylphosphate alpha-N-acetylglucosamine transferase (BOV_A0371) of which the latter may also be involved in murein biosynthesis. While it is known that B. ovis LPS has a higher affinity for antimicrobial peptides than that of B. abortus, it is unknown whether differences in LPS structure compared to smooth Brucella species affect the interaction of B. ovis LPS with toll-like receptors of the innate immune system [56,57]. Type IV secretion system (T4SS). The T4SS, encoded by virB1-virB12, is an essential virulence factor in B. abortus, B. suis, and B. melitensis [48,[58][59][60]. The genes virB1-VirB12 are intact in the B. ovis genome, suggesting that this system is functional. Further, the gene encoding its transcriptional activator VjbR (BOVA_0110; [61]) is conserved. These findings are consistent with the detection of VirB5 and VirB8 expression in B. ovis [62]. Two co-regulated genes identified as encoding T4SS substrates, vceA (BOV_1577) and vceC (BOV_1003) are present in B. ovis, however it is currently unknown whether there may be additional T4SS effectors in other Brucella spp. that are absent from B. ovis [63]. A recent report showing that B. ovis strain PA is able to replicate in macrophages and HeLa cells at a rate similar to that of B. abortus suggests that its T4SS is functional [64].
Autotransporter proteins. Four predicted autotransporters have been identified in the sequenced Brucella genomes. Each of the sequenced Brucella genomes is predicted to encode a different combination of autotransporters [21], suggesting that none of these four proteins is essential for virulence, but that different combinations of autotransporters may contribute to observed differences in tissue tropism and host preference. The B. ovis genome is predicted to encode three functional autotransporters, corresponding to B. suis BR0072, BRA0173 and BRA1148, (BOV_0071, BOV_A0152 and BOV_A1053) while the fourth gene, BOV_1937, corresponding to BR2013, is degenerate. These proteins show a range in similarity (at the amino acid level) to their B. suis counterparts, from 99% between BRA1148 and BOV_A1053, to 86% similarity between BOV_A0152 and BRA0173. BR2013, designated omaA, is a pseudogene in both B. abortus and B. melitensis, and was noted to have a polymorphism with unknown functional effects in the genome of vaccine strain B. abortus 19 [65]. Since the product of this gene has been shown to contribute to persistence of B. suis in mice [66], it is possible that lack of a functional OmaA autotransporter in B. ovis may contribute to its limited tissue tropism and host range. However, the finding that all four autotransporter genes are predicted to be pseudogenes in B. melitensis, shows that they are not absolutely required for infectivity and transmission.
Immunomodulatory functions. In addition to the T4SS, several genes implicated in modulation of the immune response by Brucella spp., are well-conserved in B. ovis ATCC25840. These include btp1, encoding the TIR domain protein, an inhibitor of TLR2 signaling, the B cell mitogen encoded by prpA, and cyclic bglucan synthase [67][68][69][70].
Outer membrane proteins. The two component regulator BvrR/BvrS, which has been shown to be a master regulator of many virulence-associated functions [71,72], appears intact in B. ovis. However, a putative envZ osmosensor (BOV_A0412) is a pseudogene. Two genes shown to encode outer membrane proteins in other Brucella species omp2a (BOV_0632 [73]) and omp31 (BOV_1565; [74]), contain point mutations in B. ovis. For Omp31, the point mutation is predicted to lead to a truncation in the protein. It was found previously that the outer membrane of B. ovis is more susceptible to cationic peptides than a rough B. abortus mutant [56], suggesting that together with the defects in LPS biosynthesis discussed above, these defects in outer membrane components may further compromise the cell envelope stability of B. ovis making it less able to survive environmental stresses.

Perspective
The unique biology of Brucella ovis compared to the human pathogenic species appears to be in part the result of genome degradation. Worldwide, the majority of human brucellosis cases occur via ingestion of contaminated dairy products. B. ovis is not known to cause human infection, despite worldwide consumption of unpasteurized sheep's milk, where B. ovis has been detected [38]. Oral transmission, although feasible in experimental conditions, does not appear to be one of the main routes of infection for B. ovis, whereas passive venereal transmission via the ewe is the most important one [75]. This suggests that this species has lost the ability to infect via the oral route. One genomic change that may contribute to this loss of oral infectivity is the loss of an important virulence factor, urease, that is required for survival of stomach acidity by Brucella spp [36,37]. Urease has also been shown to contribute to the establishment of Actinobacillus pleuropneumoniae infection in pigs through the respiratory tract [76]. If this mechanism is also operative in Brucella spp., then it is possible that B. ovis is also deficient in establishing infections not only by the digestive route but also via aerosol inhalation, which are the two main routes of infection by human pathogenic Brucella spp. Additional genes that seem to be non-functional in B. ovis could also contribute to reduce the number of transmission routes compared to other Brucella species. A characteristic of B. ovis is its tropism for the ovine male genital tract, which presents as epididymo-orchitis [8]. Since other Brucella species, especially B. melitensis are known to cause epididymo-orchitis in human patients [77], the predilection of B. ovis to cause epididymo-orchitis in rams likely represents a loss of functions required to target to other tissues. However, due to the large number of genes in the B. ovis genome with unknown function, a gain of functions that allow for increased colonization of the male genital tract cannot be ruled out based on the genome sequence.

Genome sequencing and annotation
The complete genome sequence of Brucella ovis strain ATCC25840 was determined using the whole-genome shotgun method as previously described [78]. Physical and sequencing gaps were closed using a combination of primer walking, generation and sequencing of transposon-tagged libraries of large-insert clones, and multiplex PCR [79]. Identification of putative protein-encoding genes and annotation of the genome were performed as previously described [24]. An initial set of genes predicted to encode proteins was identified with GLIMMER [80]. Genes consisting of fewer than 30 codons and those containing overlaps were eliminated. Frame shifts and point mutations were corrected or designated 'authentic.' Functional assignment, identification of membrane-spanning domains, and determination of paralogous gene families were performed as previously described [24]. Sequence alignments and phylogenetic trees were generated using the methods described previously [24].

Trinucleotide composition
Distribution of all 64 trinucleotides (3-mers) was determined, and the 3-mer distribution in 1,000-bp windows that overlapped by half their length (500 bp) across the genome was computed. For each window, we computed the x 2 statistic on the difference between its 3-mer content and that of the whole chromosome. A large value for indicates the 3-mer composition in this window is different from the rest of the chromosome (minimum of two standard deviations). Probability values for this analysis are based on assumptions that the DNA composition is relatively uniform throughout the genome, and that 3-mer composition is independent. Because these assumptions may be incorrect, we prefer to interpret high x 2 values as indicators of regions on the chromosome that appear unusual and demand further scrutiny.

Comparative genomics
The B. ovis ATCC25840 genome was compared to the genomes of B. suis 1330, B. abortus 2308, B. abortus 9-941, and B. melitensis 16M (PATRIC), at the nucleotide level by suffix tree analysis using MUMmer [81], and the predicted B. ovis CDSs were compared with the gene sets from the other sequenced Brucella genomes by BLAST and by HMM paralogous family searches, as previously described [82].

Analysis of the B. ovis-specific island
Genomic DNA from B. ovis strains and other Brucella species (B. melitensis, B. suis, B. abortus, B. canis, B. neotomae, and B. pinnipedialis) were subjected to PCR amplification of 12 target sequences within the B. ovis-specific island. PCR reactions were performed using 13 mL of a commercial PCR mix (PCR Supermix, Invitrogen, USA), 0.75 mL of a 25 mM solution of each primer (Supplementary  Table S1), and 1 mL of DNA (100 to 500 ng per reaction). Cycling parameters were denaturation at 95uC for 5 minutes; 35 cycles of denaturation (95uC for 1 min seconds), annealing (55uC for 1 min), and extension (72uC for 1 min); and a final extension at 72uC for 5 min. PCR products were resolved by agarose gel electrophoresis.

GenBank accession
The genomic sequence data are available at GenBank under accession numbers NC_009505 (Chromosome I) and NC_009504 (Chromosome II).