Brucellosis 2003 proceedings - PHIDIAS

Poster Session
bacteriological methods to the species level. In most cases it was also possible to
identify the biovars within the species.
50- THE PRODUCTION AND CHARACTERISATION OF TWO RECOMBINANT
PROTEINS, p18 AND bp26, FOR USE IN THE DEVELOPMENT OF AN
IMMUNOASSAY FOR THE DETECTION OF BOVINE BRUCELLOSIS IN SERUM
SAMPLES.
L. Dunne 1 and R. O’Kennedy 1 . (1) School of Biotechnology, Dublin City University, Glasnevin, Dublin
9, Ireland.
Brucellosis is a worldwide zoonosis caused by gram-negative, intracellular
bacteria, from the genus Brucella. In animals the disease is characterised by foetal
abortions, lowered fertility and reduced milk yields, and in humans symptoms include
undulant fever, arthritis and osteomyelitis. The disease results in economic losses
worldwide and limitations in the trade of animals and animal products internationally.
Therefore, there is an increased need for the development of rapid, sensitive and
specific methods of detection.
Currently research is focusing on the use of recombinant proteins in the
development of a highly sensitive and specific immunoassay for the detection of
bovine brucellosis. The genes encoding two recombinant proteins, an 18kDa
cytoplasmic protein (p18) and a 26kDa periplasmic protein (bp26) have been cloned
into the high level expression vector pQE60 and the proteins expressed in XL 10-
Gold E. coli. The recombinant proteins have been purified using immobilised metal
affinity chromatography (IMAC) and the suitability of the purified proteins for use in
an indirect ELISA (iELISA) established. The recombinant proteins allowed the
discrimination between Brucella-positive and negative serum samples. It is proposed
to develop an indirect immunoassay incorporating both recombinant proteins, which
will enable sensitive and specific detection of a Brucella infection in bovine serum
samples.
51- EVALUATION OF DIFFERENT CULTURE MEDIA FOR THE ISOLATION OF
Brucella suis: COMPARISON OF DIFFERENT BASAL MEDIA AND SELECTIVE
MEDIA.
A.C. Ferreira 1 , R. Cardoso 1 , D. Silva 1 , M. Silva Pereira 2 and M.I. Corrêa de Sá 1 . (1) Laboratório
Nacional de Investigação Veterinária, Lisboa, Portugal. (2) Estação Zootécnica Nacional -INIAP,
Santarérm, Portugal.
The information about the performance of Brucella suis in culture media is
scarce. These brucellae can grown in different commercial basal media but selective
media are necessary for primary isolation due to the high number of overgrowing
contaminants usually present in field samples. This study evaluates four comercial
basal media namely, Tryptic Soya Agar (TSA), Blood Agar Base (BAB), Brucella
Medium Base (BMB), GC Medium (GC) and the Plommet Medium (P) (M.Plommet,
1991). All media were used without supplements and with 5% horse serum, 1%
haemoglobin and 0.1% yeast extract (except for Blood Agar Base and Plommet
Medium). The eighteen media were tested using the viable counting techinque with
twenty-two B. suis field strains isolated from domestic pigs. Ten strains from biovar 2
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