A specific reverse complement sequence for distinguishing Brucella canis from other Brucella species

Abstract

Canine brucellosis is primarily caused by Brucella canis, but other Brucella species can also cause the disease. Identifying sequences specific to B. canis and establishing PCR assays that can distinguish between B. canis and other Brucella species is essential to determine the etiology of canine brucellosis and the source of infection and to achieve effective control. We analyzed the gaps and SNPs of genomes I and II from B. canis strain RM6/66 and B. melitensis strain 16M using the Mauve genome alignment software, and the specificity of each of these differential regions was analyzed by BLAST. A 132 bp specific sequence was found between the DK60_915 (glycosyl hydrolase 108 family protein) and DK60_917 (aldose 1-epimerase) loci in B. canis chromosome 1. Further comparative analysis revealed that this is a reverse complement sequence between B. canis and other Brucella species. Then, three primers were designed based on the sequence that could detect B. canis with a 310 bp amplification product or other Brucella species with a 413 bp product. The PCR based on these primers had reasonable specificity and a sensitivity of 100 copies of Brucella DNA. The detection results for the blood samples of the aborted dogs showed a favorable accordance with the Bruce-ladder multiplex PCR assay. In conclusion, we found a specific reverse complement sequence between B. canis and other Brucella and developed a PCR method that allows a more comprehensive identification of the pathogen involved in canine brucellosis. These findings provide an effective means for preventing and controlling brucellosis.

Keywords: B. canis; canine; canine brucellosis; reverse complementary sequence; specific.

Figure 1

The analysis of the specific sequence in B. canis. (A) BLAST result of the specific sequence. (B) The region of the specific sequence in the genome alignment. (C) The alignment of the specific sequences in B. canis and the corresponding sequence in B. melitensis. The red box represents the sequences in (D). The red sequences represent the designed primers. (D) The alignment of the specific sequence in B. canis and the corresponding reverse complement sequence in B. melitensis.

Figure 2

PCR assay based on the specific sequence. (A) Annealing temperature optimization. M, DL1000 DNA Marker; 6/66: B. canis strain RM6/66; 16: B.melitensis strain 16M. (B) Sensitivity assay of the B.melitensis strain 16M template. M, DL1000 DNA Marker. (C) Sensitivity assay of the B. canis strain 6/66 template. M, DL1000 DNA Marker. (D) Specificity assay. M, DL2000 plus DNA Marker; 1, B. canis strian RM6/66; 2, B. abortus strain A19; 3, B. suis strain S2; 4, B. melitensis strain M5; 5, B. abortus strain 2308; 6, B. suis strain 1330; 7, B. melitensis strain 16M; 8, Vanguard^® Plus 5-CVL vaccine; 9, Fel-O-Vax^® PCT vaccine (Zoetis, USA); 10, E. coli; 11, Salmonella enteritidis; 12, Shigella dysenteriae; 13, Pasteurella multocida; 14, Streptococcus hemolyticus; 15, Clostridium perfringens type C;16, Staphylococcus aureus; 17, Proteus mirabillis; 18, Candida albicans; 19, Streptococcus pyogenes; 20, Streptococcus pneumoniae; 20, Campylobacter jejuni; 21, Listeria monocytogenes; 22, Pseudomonas aeruginosa; 23, negative control.