Brucellosis is an important bacterial zoonosis caused by B. abortus and B. melitensis in Pakistan. The status of canine brucellosis caused by B. canis remains obscure. In total, 181 serum samples were collected from stray and working dogs in two different prefectures viz. Faisalabad (n = 87) and Bahawalpur (n = 94). Presence of antibodies against B. canis and B. abortus/B. melitensis was determined using the slow agglutination test (SAT) and ELISA, respectively. Real-time PCR was performed to detect and differentiate Brucella DNA at the species level. In Faisalabad, the serological prevalence was found to be 9.2% (8/87) and 10.3% (9/87) by SAT and ELISA, respectively. Only one of the ELISA positive samples (1.15%) yielded amplification for B. abortus DNA. In Bahawalpur, 63.8% (60/94) samples were found positive by SAT; however, none of the samples was positive by ELISA or by real-time PCR. Location, age (≥1 year) and body condition (weak) were found to be associated with B. canis infection, whereas presence of wounds was found to be associated with B. abortus infection only. These findings point towards a risk of transmission from dog to livestock and humans and vice versa. The study expects to draw the attention of concerned authorities towards infection prevention and animal welfare. This study warrants further epidemiological investigation on brucellosis in pet dogs and their owners. To the best of our knowledge, this is first ever report on B. canis and B. abortus in dogs in Pakistan
