An imported dog was confirmed to be positive with canine brucellosis in Sweden in 2010. The whole genome of Brucella canis SVA10 was subjected to phage analysis (WGS-PA) and was assigned to the Asian B. canis cluster. Further analysis indicated that the genome of B. canis SVA10 is smaller compared to genomes of the same species. A 35,781 bp genomic island (GI) was found to be absent in strain SVA10 which was detected by read mapping the paired reads to the genome of B. canis ATCC 23,365 T . The lacking genes of genomic island GI FeGSH are mainly coding for iron uptake enzymes and parts of the glutathione pathway. A screening of all available whole genome sequences of Brucella strains confirmed that GI FeGSH is also missing in four more strains of B. canis but present in several strains of B. abortus, B. melitensis, B. suis, B. ovis, B. microti , B. pinnipedialis , and B. ceti . Parts of the GI were present, but scattered in two other B. canis strains. The aim of this study was to find differences in the genomes of Brucella which might explain former described differences in virulence. The analysis was extended to all available Brucella genomes after the detection of a genomic island in strain SVA10.
The genus Brucella currently consists of 11 species of which B. melitensis, B. abortus, and B. suis are further classified in different biovars. The biovar concept is useful especially regarding epidemiological source tracing queries. However, the genetic divergence within the whole genus Brucella is very low and makes genotyping challenging. Phylogenetic analyses based on 16S rDNA commonly used for bacterial speciation are not possible with the Brucella species since they share 100% identical 16S rRNA genes. Comparing the whole genomes of all Brucella species with DNA-DNA hybridization, the similarity is still between 87% and 100% [
Brucella canis infects dogs and humans and is together with B. melitensis, B. abortus, and B. suis one of the more dangerous Brucella species regarding zoonotic potential, infectious dose and global distribution. Sweden is officially free of brucellosis and there has only been one case [
The aim of this study was a detailed investigation of the genome of the B. canis strain SVA10, including screening for genomic variations as well as the comparison of the SVA10 genome to other Brucella species genomes regarding potentially genes which might explain differences in Brucella virulence.
Brucella canis strain SVA10 was isolated at the National Veterinary Institute of Sweden from an American Staffordshire terrier imported from Poland [
A validated workflow for sequencing and analysis of bacterial samples was chosen for whole genome sequencing (WGS). The DNA of B. canis strain SVA10 was extracted from cultivated colonies using an EZ-1 extraction robot and EZ-1 DNA tissue kit (Qiagen, Hilden, Germany). The libraries were prepared with a Nextera XT sample preparation kit (Illumina, San Diego, CA) which allows to proceed samples with a DNA concentration of 0.5 ng/µl. WGS was performed using a 2 × 300 paired-end run on an Illumina MiSeq platform (San Diego, California, USA).
The reads were de novo assembled using the Mira plugin version 1.0.1 in Geneious version 8.1.7 [
B. canis strain | NCBI Accession number | Origin | Presence of GIFeGSH | WGS-PA cluster | ||
---|---|---|---|---|---|---|
Source | Collected | Geographical | ||||
ATCC 23365T | NC_010103 | dog | 1968 | n.a. | + | AAE |
RM6/66T | CP007758 | dog | 1968 | n.a. | + | AAE |
SVA13 | CP007629 | dog | 2013 | Sweden/Spain | + | AAE |
Oliveri | HG803175 | dog | n.a. | Columbia | + | AAE |
04-2330-1 | AXNG01000001-004 | n.a. | n.a. | n.a. | + | AAE |
96-7258 | AXNF01000001-004 | n.a. | n.a. | n.a. | + | AAE |
CNGB 1324 | AQMZ01000001-002 | human | 2008 | Argentinia | + | AAE |
CNGB 513 | AQJZ01000001-007 | human | 2001 | Chile | + | AAE |
CNGB 1172 | AQMY01000001-002 | human | 2006 | Columbia | + | AAE |
F7/05A | AQNA01000001-003 | dog | 2005 | South Africa | − | AAE |
HSK A52141 | CP003174 | dog | ~2011 | Korea | +/− | AAE |
SCL | LGAQ00000000.1 | dog | 2008 | Chile | +/− | A |
UK10/02 | AQNB01000001-002 | n.a. | 2002 | n.a. | + | A |
79/122 | AQJY01000001-005 | dog | 1979 | Japan | − | A |
118 | AMOZ01000001-154 | human | 2008 | China | − | A |
BCB018 | ALOJ02000001-170 | human | 1988 | China | − | A |
SVA10 | MAXW00000000 | dog | 2010 | Sweden/Poland | − | A |
+: GI is present; −: GI is absent; +/−: GI is scattered/incomplete, n.a.: no data available.
The reads were assembled into 12 contigs which were deposited in the NCBI database with the accession numbers MAXW00000000, Bioproject PRJNA328097 and Biosample SAMN05363686.
The range of the genome size of B. canis is 3,217,060 bp (strain F7/05A) to 3,318,660 bp (strain Oliveri). The genome of B. canis strain SVA10 consists of 3,264,482 bases with a G+C content of 57.24%. The examined strain has one of the smallest genomes of all available sequenced B. canis strains. In addition to the de novo assembly, the sequence reads of strain SVA10 were mapped against the whole genome sequence of the type strain of the species (ATCC 23,365T; 3,312,769 bases) with the aim to detect the lacking genes of the missing 48,287 bases. The main differences between the two strains regarding lacking genes were observed at chromosome II (
Two major systems, coding for a Fe3+ uptake system and a glutathione pathway were a striking feature on GIFeGSH (
The second predominant cluster of genes that are not present in the genome of B. canis strain SVA10 are genes that are regulating the glutathione pathway (EC:1.8.1.12, EC:3.4.11.2, EC:6.3.1.9, EC:3.5.2.9). Glutathione is an antioxidant and uptake and utilization systems are present in several bacterial species [
Gene | Length bp |
---|---|
ABC transporter permease | 870 |
Adenine deaminase | 1800 |
Adenine permease | 1293 |
Aldehyde dehydrogenase | 1446 |
Amino acid ABC transporter substrate-binding protein | 774 |
Aminotransferase | >16 |
Aminotransferase | 855 |
Branched-chain amino acid ABC transporter | 717 |
Branched-chain amino acid ABC transporter permease | 1000 |
Cytidine deaminase | 600 |
Enterobactin ABC transporter permease | 954 |
FAD-binding dehydrogenase | 1656 |
Glycerol-3-phosphate transporter membrane protein CDS; sn-glycerol 3-phosphate ABC transporter permease | 849 |
Glycerol-3-phosphate transporter permease CDS; sn-glycerol 3-phosphate ABC transporter permease | 875 |
Guanine permease | >778 |
Guanine permease | >449 |
Hemolysin III | 750 |
Insertase | >11 |
Iron ABC transporter ATP-binding protein | 759 |
Iron ABC transporter permease | 963 |
Iron ABC transporter substrate-binding protein | 970 |
Leu/Ile/Val-binding protein homolog 6 CDS; amino acid-binding protein | 1173 |
Membrane protein | 762 |
Membrane protein | 489 |
Peptidase | 1041 |
Permease CDS | 1772 |
Ribonuclease P CDS; ribonuclease P protein component | >11 |
Sn-glycerol-3-phosphate-binding periplasmic protein UgpB | 1302 |
Sugar ABC transporter ATP-binding protein | 1056 |
Sugar ABC transporter permease | >10 |
Transcriptional regulator | 609 |
Transporter | 918 |
in a few strains might explain why some B. canis strains are more virulent than others and why they may differ in zoonotic potential. Additional genes related to bacteria’s virulence that were found on GIFeGSH are Hemolysin III and Peptidase.
The necessity of additional genomic content in B. canis is disputable since obligate intracellular as well as facultative intracellular pathogens show a trend to reduce their genomes during evolution [
Not much is known about the epidemiology of human infections caused by B. canis [
The detection of GIFeGSH which contains potential virulence genes gives the possibility to assess the effect to the host, the possible transmission as well as the consequences of a Brucella infection. Curing brucellosis caused by bacteria containing GIFeGSH is expected to be more challenging and the bacteria might affect the host cells with a higher efficiency.
GIFeGSH might be applicable for source tracing if a strain contains unique scattered parts of GIFeGSH in their genomes as the strains HSK A52141 and SCL (
Due to the high genetic homology it is difficult to find epidemiological markers within the genomes of several Brucella species. A new whole genome sequenced strain of Brucella canis (SVA10) which was derived from an infected dog imported from Poland to Sweden was analysed and assigned to the Asian WGS-PA cluster [
We thank Joakim Ågren for supportive discussions in bioinformatics. This work was supported by the Swedish Civil Contingencies Agency (MSB).
The authors declare that there is no conflict of interest.
Wahab, T., Skarp, A., Båverud, V. and Kaden, R. (2017) GIFeGSH: A New Genomic Island Might Explain the Differences in Brucella Virulence. Open Journal of Animal Sciences, 7, 141-148. https://doi.org/10.4236/ojas.2017.72012