Abstract
Bovine brucellosis induces abortion in cows, produces important economic losses, and causes a widely distributed zoonosis. Its eradication was achieved in several countries after sustained vaccination with the live attenuated Brucella abortus S19 vaccine, in combination with the slaughtering of serologically positive animals. S19 induces antibodies against the smooth lipopolysaccharide (S-LPS), making difficult the differentiation of infected from vaccinated bovines. We developed an S19 strain constitutively expressing the green fluorescent protein (S19-GFP) coded in chromosome II. The S19-GFP displays similar biological characteristics and immunogenic and protective efficacies in mice to the parental S19 strain. S19-GFP can be distinguished from S19 and B. abortus field strains by fluorescence and multiplex PCR. Twenty-five heifers were vaccinated withS19-GFP (5×109 CFU) by the subcutaneous or conjunctival routes and some boosted with GFP seven weeks thereafter. Immunized animals were followed up for over three years and tested for anti-S-LPS antibodies by both the Rose Bengal test and a competitive ELISA. Anti-GFP antibodies were detected by an indirect ELISA and Western blotting. In most cases, anti-S-LPS antibodies preceded for several weeks those against GFP. The anti-GFP antibody response was higher in the GFP boosted than in the non-boosted animals. In all cases, the anti-GFP antibodies persisted longer, or at least as long, as those against S-LPS. The drawbacks and potential advantages of using the S19-GFP vaccine for identifying vaccinated animals in infected environments are discussed.
Highlights
Brucellosis, caused by species of the genus Brucella is a widespread disease that affects a great variety of domestic and wildlife hosts, including humans [1]
Brucella strains were routinely grown in Blood Agar Base N 2 (BAB; Biolife), trypticase soy broth (TSB), and E. coli strains on Luria Broth (LB) either plain or supplemented with 100 μg/mL ampicillin, 35 μg/mL kanamycin, 5 μg/mL nalidixic acid, 15 μg/ml gentamicin, or 1 mg/mL erythritol, all from Sigma-Aldrich
We showed that an S19 construct constitutively expressing green fluorescent protein (GFP) (S19-GFP) maintains the biological and immunological properties as the parental S19 reference vaccine strain
Summary
Brucellosis, caused by species of the genus Brucella is a widespread disease that affects a great variety of domestic and wildlife hosts, including humans [1]. Vaccination of cattle with S19 combined with adequate diagnostic testing and culling of the seropositive animals (known as “test and slaughter” programme), has been instrumental to eradicate bovine brucellosis from many countries [1, 3, 7,8,9,10,11,12]. S19 induces residual antibodies against the Brucella N-formyl perosamine homopolysaccharides that built the O-chain of the smooth lipopolysaccharide (S-LPS) and native hapten (NH) polysaccharides, the main antigens used in the diagnosis of brucellosis. This can cause positive reactions in serological tests that use these polysaccharides as antigens [3, 13, 14]. Despite the existence of vaccination strategies and diagnostic tests capable to differentiate infected from S19 vaccinated animals [10,11,12, 14,15,16], the S19 vaccine was banned in the United States [17] and its application abandoned in many other countries [3, 18]
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