Indian Journal of Animal Sciences 92 (11):1274–1279, November 2022/Article https://doi.org/10.56093/ijans.v92i11.124795 Molecular characterization of Brucella species detected from clinical samples of cattle and buffaloes VARSHA THORAT 1* and ANILKUMAR BANNALIKAR 1 Mumbai Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur, Maharashtra 400 012 India Received: 22 June 2022; Accepted: 25 August 2022 ABSTRACT The present study was undertaken for molecular characterization of Brucella species of cattle and buffaloes. Clinical samples (1145) of unvaccinated cattle and buffaloes (200 blood samples, 710 sera, 190 vaginal swabs, 20 abomasal contents of foetus, 25 foetal tissues) and 146 blood samples of vaccinated animals were collected from dairy farms in and around Mumbai and Pune region. These samples were processed for isolation of Brucella organisms and further characterized by PCR and sequencing. A total of 26 (11.06%) Brucella isolates were recovered from 235 samples. Also, 5 isolates received from human cases were included in the study. BCSP 31 PCR showed an amplicon of 223 bp in all 31 isolates, 123 (61.5%) blood samples, 123 (64.73%) vaginal swabs and 27 (60%) aborted foetal material. IS711/AB and BM PCR showed an amplicon of 498 bp and 731 bp in 17 and 14 isolates, 42 (21%) and 38 (19%) blood samples, 43 (22.63%) and 34 (17.89%) vaginal swabs, while 7(15.55%) and 6 (13.33%) aborted foetal material, respectively. The phylogenetic analysis detected the ancestral origin of the organism. Rapid and correct diagnosis of brucellosis and vaccination is important to eradicate the disease. The molecular methods used in the present study speed up the diagnosis of the disease. Keywords: Brucella abortus, B. abortus 544, BCSP 31, IS711 PCR assays Present address: 1 Department of Veterinary Microbiology, Mumbai Veterinary College, Maharashtra Animal and Fishery Sciences University, Nagpur, Maharashtra. * Corresponding author email: varshamicrovet@gmail.com Brucellosis is a vital disease of cattle and buffaloes that causes infertility in male and female animals. The disease causes severe economic losses to the livestock industry by reducing the productive and reproductive potential of the affected animals. The disease remains neglected and results in major health, economic and livelihood burden due to ineffcient control methods (Deka et al. 2018). It is a zoonotic infection, so humans may get the disease condition by contacting infected animals. Abortions are generally seen in cattle and buffaloes in the last trimester of pregnancy due to brucellosis. Infections in female animals may also cause stillborn or weak calves, retained placentas and reduced milk yield. In male animals, seminal vesicles, ampullae, testicles, and epididymides may be infected, and occasionally, the bacteria localizes in the joints causing arthritis (Shakuntala et al. 2021). Brucellosis is often diagnosed by serology and culture. The detection of microbes from the tissue samples, milk or vaginal exudates followed by bacteriological characterization is considered as the ‘gold standard’ (Surucuoglu et al. 2009). But these approaches demand a high level of expertise to isolate the organism; they are time-consuming and involve the risk of laboratory-acquired infection (Shome et al. 2019). The serological diagnosis of brucellosis is relatively simple, inexpensive and widely used to diagnose in animals and human. However, the major limitation of this procedure is its doubtful specifcity. The nucleic acid-based detection methods developed in recent times are promising tools for diagnosing brucellosis. Amplifcation of DNA by PCR is currently used to diagnose several infectious diseases caused by fastidious or slowly growing bacteria. These techniques are sensitive, specifc, quick and inexpensive. Additionally, the methods do not require the handling of living organisms, thereby reducing the safety concerns. Several targets have been explored to fnd out their suitability in identifcation and typing. Some of the targets that have been evaluated extensively include 16S rRNA, insertion sequence IS711 and Brucella Cell Surface Protein (Mittal et al. 2018). Considering all these aspects, the present study was planned for molecular characterization of Brucella spp. from clinical samples of cattle and buffaloes. MATERIALS AND METHODS Permission was taken from Institutional Biosafety Committee for conducting research as per letter ref No.BVC/Dean/VPH/IBSC/221/2016 dated 02/07/2016. Reference strains: Reference strains of Brucella, i.e. Brucella abortus 544, Brucella melitensis Rev 1 and Brucella abortus S-19 were purchased from Division of SUPPLEMENTARY MATERIAL AVAILABLE ONLINE 22