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Evaluation of Molecular Methods for the Detection of Brucella Species in Water Buffalo Milk

C. Marianelli^*,1, A. Martucciello, M. Tarantino^*, R. Vecchio, G. Iovane and G. Galiero

^* Dipartimento di Sanità Pubblica Veterinaria e Sicurezza Alimentare, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Sezione Diagnostica Provinciale di Salerno, Centro di Referenza Nazionale sull’igiene e le tecnologie dell’allevamento e delle produzioni bufaline, Via delle Calabrie 27, 84132 Fuorni-Salerno, Italy
Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Della Salute 2, 80055 Portici-Napoli, Italy

¹ Corresponding author: cinzia.marianelli{at}iss.it

Brucellosis is a highly infectious disease affecting both animals and humans. The current standard tools for the diagnosis of this bacterial infection are serological and microbiological. In this study, we evaluated the feasibility of molecular assays as diagnostic tools for the detection of Brucella spp. in water buffalo milk. For this purpose, we first compared different DNA extraction protocols and PCR methods on artificially spiked milk samples. The most sensitive methods were then used to examine milk from serologically positive and negative water buffaloes. Molecular results were compared with serological and bacteriological test results. Milk samples from 53 Brucella seropositive buffaloes (by either rose Bengal or complement fixation test) were positive by ELISA, 37 were positive by culture, 33 were positive by PCR, and 35 were positive by real-time PCR. Of the 37 culture-positive samples, a total of 25 and 26 were positive by PCR and real-time PCR, respectively. Of the 16 culture-negative samples, 8 were positive by PCR and 9 by real-time PCR. Thus, although culture showed greater sensitivity than PCR, some animals found positive by serological methods and PCR tested negative by milk culture. The combined use of bacteriological and molecular tools increased the number of positive samples to 46. In conclusion, these results suggest that the simultaneous application of these 2 direct detection methods (culture and PCR) could be more useful than one test alone for the diagnosis of Brucella spp. in buffalo milk.

Key Words: Brucella spp. • buffalo milk • DNA extraction • polymerase chain reaction