Brucellosis 2003 proceedings - PHIDIAS

Poster Session
Brucella ovis, Brucella canis, Brucella neotomae) instead of time consuming
biochemical test. The purpose of this study was to compare biochemical test and
PCR-RFLP to investigate the prevalence of Brucella canis in dogs in the future. A
total of 260 dogs were randomly selected from two different breed kennels, (1) one
kennel that brucellosis has occurred (group 1, 126 dogs from 6 farms), and (2)
random selected breed kennel (group 2, 134 dogs from 6 farms). Of 126 dogs in
group 1, 47 dogs (37.3%) were bacterial culture positive, while 5 dogs (3.7%) were
culture positive in group 2. Brucella canis was isolated from all 6 farms in group 1,
while Brucella canis was isolated from 2 farms in group 2. To characterize and
differentiate Brucella canis isolates from Brucella melitensis, Brucella suis, Brucella
abortus, omp-31, wbkA, and per were amplified and treated with restriction enzyme.
Omp-31 was amplified from all Brucella species, except B. abortus. PCR-RFLP
analysis of omp-31 revealed that all Brucella canis showed species specific B type
following digestion with Bme18I. However, all the Brucella strains showed the same
pattern following treatment with SalI. PCR-RFLP analysis of wbkA revealed that all
the Brucells spp. showed the same pattern (A type) following digestion with HindIII.
PCR-RFLP analysis of per revealed that Brucella canis and Brucella suis showed
same pattern following treatment with HindIII. However, B. abortus 544 and Brucella
melitensis 63/9 showed different pattern. Our results showed that PCR-RFLP of omp-
31 can be applicable to investigate the prevalence of Brucella canis.
49- MOLECULAR CHARACTERISATION OF FIELD STRAINS OF Brucella sp.
ISOLATED IN ITALY IN THE YEARS 2001-2003.
M. Ancora 1 , P. De Santis 1 , E. Di Giannatale 1 , T. Persiani 1 , A.P. MacMillan 2 and V. Caporale 1 . (1)
Istituto Zooprofilattico Sperimentale dell‚Abruzzo e del Molise “G. Caporale”, Italy. (2) Central
Veterinary Laboratory, Weybridge, United Kingdom.
Classically the genus Brucella is deemed to comprise six species: B.
melitensis, B. abortus, B. ovis, B. canis, B.suis and B. neotomae, these subdivided
further into biovars or biotypes. On the basis of the homology shown during DNA-
DNA hybridisation studies, it has been suggested that Brucella is a monospecific
genus represented by the single species B. melitensis comprising various infraspecies
considered as biovars. Some databases and culture collections (GenBank;
National Culture Collection, UK) have accepted this scheme, but other authors, on
the basis of host range and species-specific outer membrane protein gene markers,
etc, prefer to retain the six Brucella species as biologically separate entities. In our
study we applied molecular methods in the routine identification of field strains of
Brucella isolated in Italy between 2001-2003. A PCR method, based on the insertion
sequence IS711, is used to verify that a field isolate is a Brucella, and then a second,
more refined, species-specific PCR method, based on published data, is used to
identify an isolate to the species level. The different Brucella species were finally
analysed by RFLP, using prior amplification, by PCR, of the omp2a, omp2b and
omp25 genes, and subsequent restriction analysis of the amplicons with selective
restriction enzymes to finally assign the Brucella species to the correct biovars. All
245 Brucella field isolates have been processed; 152 were shown to be B. melitensis
biovar 3, 23 B. abortus biovar 1, 39 B. abortus biovar 6, 3 B. ovis, 30 B. suis biovar 1,
and 1 B. suis biovar 2. For all the field isolates screened by the methods described,
there was complete agreement with the identifications made by conventional
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Brucellosis 2003 International Research Conference