| Summary: | Brucellosis is a worldwide zoonosis causing important economic loss and a public
health problem. Small ruminants are the preferred hosts of Brucella melitensis and
thus the main source of human infections. Effective control of sheep and goat brucellosis has been achieved in several countries through vaccination with the live‐
attenuated B. melitensis Rev1 vaccine. However, Rev1 induces a long‐lasting serological response that hinders the differentiation between infected and vaccinated
animals. A Rev1::gfp strain expressing constitutively the Green Fluorescent Protein
(GFP) was built by stable insertion of a mini‐Tn7‐gfp in the glmS-recG non‐codifying
chromosomal region. An associated indirect ELISA‐GFP was developed to identify
anti‐GFP antibodies in vaccinated animals. The resulting Rev1::gfp kept the biological properties of the Rev1 reference strain, including residual virulence and efficacy
in mice, and was readily distinguished from Rev1 and other Brucella field strains by
direct visualization under ultraviolet illumination, fluorescence microscopy and a
multiplex PCR‐GFP. The Rev1::gfp strain did not elicit anti‐GFP antibodies itself in
lambs but when applied in combination with recombinant GFP induced an intense
and long‐lasting (>9 months) anti‐GFP serological response readily detectable by the
ELISA‐GFP. Overall, our results confirm that Rev1 GFP‐tagging can be a suitable
alternative for identifying vaccinated sheep in infected contexts.
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