Multiple-locus variable number of tandem repeat analysis assay as a tool for the epidemiological study of Brucella canis in Korea

* Correspondence: [email protected] Brucella canis is an etiological agent of canine brucellosis, a zoonotic disease that can be transmitted to humans. B. canis is associated with dogs, which are its natural host. In some cases, dogs can also be infected with other Brucella species such as B. abortus, B. melitensis, and B. suis; however, B. canis is the primary etiological agent responsible for canine brucellosis (1,2). The clinical symptoms of canine brucellosis are abortion in late pregnancy, epididymitis, and prostatitis, all of which can cause reproductive failure (2,3). Thus, canine brucellosis can result in economic losses with respect to kennels. Transmission among dogs occurs while mating or through direct contact with vaginal discharge after abortion, infected placenta, or fetus. Infections can also be transmitted through urine, since B. canis can be shed in the urine for several years (2–4). Therefore, brucellosis can be easily transmitted among dogs living in close proximity. Although B. canis infection in humans is rare, it can occur through exposure to contaminated canine tissues or fluids such as products exuded during conception and abortion or vaginal discharge. B. canis infection in humans can be difficult to diagnose because of the nonspecific disease symptoms such as undulant fever, headache, and weakness (5,6). Therefore, identification of the source of infection, treatment, and eradication of canine brucellosis may be an effective treatment strategy. Many methods have been developed for molecular subtyping in the last decade and multiple locus variable number of tandem repeat analysis (MLVA) has proven to be highly appropriate for analyzing phylogenetic relationships among isolates and epidemiological trace-back investigations (7,8). MLVA has been used in epidemiological studies of Brucella species (9,10), Streptococcus agalactiae (11), Pseudomonas aeruginosa (12), Bacillus anthracis (13,14), and Salmonella typhimurium (15,16). The discriminatory power of the assay has been increased by the inclusion of more markers. However, MLVA has not been commonly used in epidemiological studies with B. canis. In this study, MLVA-18 was employed to analyze B. canis isolates from different areas and trace the infection source. Blood of a 1-month-old domestic dog was sent to the diagnostic laboratory for bacteriological testing, asking especially for B. canis. The serological test was positive (data not shown) and B. canis was isolated from blood culture. Following this, an epidemiological survey was conducted, which traced the source of infection to a Yangju-si breeding farm, which was also the location of the domestic dog with brucellosis. The epidemiological survey also identified Namyangju-si breeding farm as the birthplace of the infected domestic dog, after which the dog was moved to the Yangju-si breeding farm, which was the location of its adoption. All the dogs from the two breeding farms were Abstract: Multiple-locus variable number of tandem repeats analysis (MLVA) is useful for epidemiological investigations. In this study, MLVA-18 was used to assess the genetic and epidemiological relationships of Brucella canis isolates among a domestic dog and dogs from two breeding farms. MLVA-18 assay showed variation in copy numbers in 6 of the 18 loci. Interestingly, the YJ02-1 strain isolated from the pet dog from Yangju-si showed only one tandem repeat change in Bruce 09 locus as compared with that of NYJ-1 strain isolated from a dog from the Namyangju-si farm. The two strains were shown to have originated from the same source or a closely related strain.