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...