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Brucella-specific nucleotide sequences encoding the BCSP 31 kDa protein, Omp2 and the 16S rRNA were employed in three independent diagnostic PCR assays. Results of the three PCR assays on six reference strains of Brucella were in complete agreement. The results of PCR assays based on bcsp and omp2 on 19 Indian field isolates (human, bovine and murine tissues) also agreed completely. However, when the 16S rRNA gene was employed as the diagnostic target in the PCR, only 14 out of these 19 isolates and 2 out of 7 bovine milk isolates were identified as the genus Brucella. The bovine blood samples were insensitive to 16S rRNA PCR. The antibody-detecting ELISA results of field samples (n587) from a serologically positive herd in India were compared separately with omp2 and bcsp PCRs of blood (n562). While the bcsp PCR was the most sensitive, the degree of association of ELISA with omp2 blood PCR (k50.37 at P ,0.05) was similar to that with the bcsp blood PCR (k 50.34 at P ,0.05). An improvement in the correlation between ELISA and blood PCR was noticed (k 50.5 at P ,0.05) when a consensus result of omp2 and bcsp blood PCR was considered for comparison with ELISA. The use of more than one marker-based PCR gave increased sensitivity and higher specificity and appears to be a more reliable molecular diagnostic approach for screening of field animals. INTRODUCTIONBrucellosis causes infertility and abortion in bovines (Radostits et al., 1994;Corbel, 1997) and undulant fever in humans (Corbel & Brinley-Morgan, 1984). Bovine brucellosis is usually caused by Brucella abortus, and less frequently by Brucella melitensis and Brucella suis (OIE, 1996). Among the six recognized species of Brucella, B. abortus, B. melitensis, B. suis and Brucella canis can potentially infect humans (Nicoletti, 1980) while Brucella ovis and Brucella neotomae have not been isolated from humans.Accurate diagnosis of brucellosis requires bacteriological isolation and detection of the pathogen in the laboratory, which is impractical for regular screening of large populations (Alton et al., 1988;Lulu et al., 1988;Radostits et al., 1994;Yagupsky, 1994). Serological tests can be nonspecific owing to cross-reaction or subsensitive or high immunity reactions, depending on subclinical or endemic prevalence of the disease (Ariza et al., 1992;Weynants et al., 1996;Godfroid et al., 2002). Numerous PCR-based assays have been developed for the identification of the genus Brucella from cultures, animal/human tissues and animal products. These employ the gene encoding the 31 kDa Brucella cell surface salt extractable protein (BCSP), omp2, 16S rRNA, IS711 and other gene targets (Baily et al., 1992; Leal-Klevezas et al., 1995;Da Costa et al., 1996;Rijpens et al., 1996;Bricker, 2002; Morata et al., 2003;Bogdanovich et al., 2004;Mukherjee et al., 2005;O'Leary et al., 2006). Real-time PCRs for high sensitivity detection (Redkar et al., 2001; Probert et al., 2004;Navarro et al., 2006;Queipo-Ortuno et al., 2005) and differential/multiplex PCRs for strain typing based on...

This study was carried out to investigate the prevalence of bovine brucellosis and infectious bovine rhinotracheitis (IBR) in organized dairy farms with history of abortion in India. ELISA and Rose Bengal Plate Test (RBPT) were used to detect the seropositive animals and the test results indicated that 22.18% and 13.78% animals were declared as sero-positive by ELISA and RBPT, respectively. Milk Ring Test (MRT) was carried out only in one farm and 12.82% of the tested animals were turned positive. Culture examination analysis of milk samples, two animals revealed the presence of organisms indistinguishable from Brucella spp. The organism was confirmed as brucella by morphological characteristics and biochemical tests. An overall sero-prevalence of antibodies against IBR was found to be 60.84%. None of the genital and nasal swab samples was found to be positive for presence of bovine herpesvirus -1 (BHV-1) on repeated passage in Madin-Darby Bovine Kidney (MDBK) cell lines. Brucella and IBR considered as the causal agent for abortions in these farms. The present study indicates the urgent need and the necessity for control of these infectious diseases which cause heavy economic losses to the organized farms.

The majority of tuberculosis cases in ruminants are caused by Mycobacterium bovis (MB). However, in this study, the authors reported the isolation of Mycobacterium tuberculosis (MT) from bovine milk, nasal swabs and post-mortem tissue samples (n = 841) collected from cattle and buffaloes in the states of Telangana, Maharashtra and Gujarat in India in the period from 2010 to 2015. The isolates (n = 7) were confirmed as Mycobacterium due to their growth characteristics and colony morphology in a commercial liquid medium Mycobacteria Growth Indicator Tube (MGIT)™ employing the BD BACTEC™ MGIT™ 960 system and the Lowenstein Jensen (LJ) medium supplemented with glycerol but not with sodium pyruvate, and BD-DIFCO™ Middlebrook 7H10 agar containing oleic albumin dextrose catalase (OADC). These isolates were initially identified as members of the Mycobacterium tuberculosis complex (MTC) using a commercial nested polymerase chain reaction (PCR) kit based on the IS6110 MTC specific nucleotide sequence. The isolates were confirmed as MT using three commercial line probe assay kits, were further genotyped, and the spoligotypes identified were of East African Indian (EAI) 3_IND, EAI5, Central-Asian (CAS) 1_DELHI, U and T1 lineages. Two MT isolates from one antelope (Antelope cervipara) and one gazelle (Gazelle bennettii) from Gujarat, which were identified previously, were spoligotyped during this study and identified as belonging to EAI3_IND and EAI5 lineages, respectively. The epidemiological significance and zoonotic implications of regional presence and documentation of the same or two different spoligotypes in different species within the family Bovidae as well as humans is discussed.