20 Listeriosis, an important food-borne infection, is caused by Listeria monocytogenes. Till date, 13 serovars have been described for the species L. monocytogenes, at least 95% of the strains isolated from foods and patients were of serovars 1/2a, 1/2b and 4b (Graves et al. 1999). Various reports indicate that Listeria spp. can be found in dairy products (Marth and Ryser 1990), but the outbreaks associated with the consumption of milk (Brito et al. 2008) are of great concern in the dairy industry due to high mortality rate. One of the reasons for infection via dairy products could be attributed to the ability of L. monocytogenes to survive and grow over a wide range of temperature and low pH (Gahan et al. 1996). The pathogen was found involved in numerous outbreaks of listeriosis involving the consumption of milk and milk products (Jamali et al. 2013, Ning et al. 2013). India, being the largest producer of milk in the world, also has the highest number of cattle in the world. During the year 2011–12, the estimates of milk production revealed the milk production in Punjab as 9.55 million tonnes versus 127.904 million tonnes for India (Source: Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, Present address: 1 (dr.laximansawant@gmail.com), School of Public Health and Zoonoses. Indian Journal of Animal Sciences 86 (5): 512–517, May 2016/Article Molecular characterization of Listeria monocytogenes in bovine milk and evaluating the sensitivity of PCR for direct detection in milk SAWANT LAXIMAN 1 , SIMRANPREET KAUR 2 , R S AULAKH 3 and J P S GILL 4 Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab 141 004 India Received: 24 July 2015; Accepted: 14 September 2015 ABSTRACT Food-borne listeriosis, recognized as an emerging bacterial disease of humans and animals worldwide, is caused by L. monocytogenes with at least 95% of the strains isolated from foods and patients belonging to serovars 1/2a, 1/ 2b and 4b. Milk and dairy products were implicated as sources of listeriae in several widely publicized incidents, thus suggesting that the mammary glands of mastitic cattle may be an important reservoir of Listeria. In the present study, 350 bovine milk samples were collected for prevalence and molecular characterization studies of Listeria spp. The isolates were phenotypically and genotypically characterized by biochemical tests, haemolysis on sheep blood agar, CAMP test, PI-PLC assay and multiplex PCR targeting virulence cluster genes namely haemolysin (hlyA), PI-PLC (plcA), actin (actA), p60 (iap) and regulatory (prfA); along with multiplex PCR for typing major serovars targeting lmo0737, ORF2819, ORF2110 and prs genes. Four pathogenic L. monocytogenes were recovered indicating prevalence rate of 1.14% in milk while the overall prevalence rate of Listeria spp. was 1.42%. All the four pathogenic isolates were characterized as L. monocytogenes serotype 4b. Antibiogram of the pathogenic L. monocytogenes isolates revealed sensitivity for amikacin, gentamycin, norfloxacin and doxycyclin. Animal sera (169) screened by indirect ELISA for antibodies against listeriolysin O showed sero-positivity of 7.1%. Sensitivity of PCR for direct detection from milk was evaluated to be 8.8 × 10 5 L. monocytogenes cells/ml of milk. Thus, the presence of pathogenic strains of L. monocytogenes in raw milk appeared to be a cause for concern with profound public health implications. Key words: Antibiogram, Bovine, ELISA, Listeria monocytogenes, Listeriosis, Milk, Sensitivity, Serotype Government of India). Various workers in India have also reported the isolation of pathogenic L. monocytogenes from milk (Bhilegaonkar et al.1997, Barbuddhe et al. 2002, Rawool et al. 2007). Current microbiological culture methods require at least 5 days to isolate Listeria colonies from food matrices and additional days for the identification at the species level, and are therefore time-consuming and laborious (Rapporti ISTISAN 1996). PCR-based detection systems, which are specific and sensitive, were thus proposed to eliminate the enrichment and culturing step (Makino et al. 1995). The present study, therefore is aimed to undertake the phenotypic and molecular characterization of L. monocytogenes isolates identified from milk samples of cows and buffaloes in Punjab state of Northern India and to evaluate the sensitivity of PCR for direct detection of L. monocytogenes in milk, thereby eliminating the need for enrichment and subsequent culturing on plating media. MATERIALS AND METHODS Bacterial cultures: The standard strains of Listeria monocytogenes 4b (ATCC 19115), Staphylococcus aureus (ATCC 11632) and Rhodococcus equi (ATCC 6939) were used in this study. Samples: Bovine milk samples (350) were collected