Avaliação de frações hidrofóbicas e hidrofílicas de brucella abortus em ensaios imunoenzimáticos para caracterizar o perfil de anticorpos produzidos por bovinos vacinados e não-vacinados

Abstract

Brucella abortus hydrophobic and hydrophilic fractions obtained from smooth lipopolysaccharide (S-LPS) and Triton X-114 extractions, respectively, were evaluated in immunoassays in order to characterize the antibody response of B. abortus S19 vaccinated heifers and non-vaccinated seropositive cows. Indirect enzyme immunoassays (iELISAs) using Protein A or anti-bovine IgG as peroxidase conjugates as well as immunoblot and avidity-immunoblot were used. Four groups with 15 cattle sera each were analyzed: (I) non-vaccinated seropositive cows from Brucella-endemic areas; (II) non-vaccinated seropositive cows from Brucella non-endemic areas; (III) S19 vaccinated heifers from Brucella-endemic areas; and (IV) non-vaccinated seronegative cows from Brucella non-endemic areas. Traditional agglutination tests were compared to iELISAs. A threshold ELISA index (EI) value for a result to be considered positive was selected through two-graph receiver operating characteristic (TG-ROC) analysis. iELISAs were more able to identify vaccinated heifers as negative animals (87%) in relation to classical agglutination tests (13%). Levels of IgG antibodies to B. abortus S-LPS were higher in non-vaccinated seropositive cows (groups I and II) when Protein A/peroxidase was used, reflecting in predominance of IgG2 antibodies. Avidity-immunoblot with B. abortus Triton X-114 hydrophilic fraction showed significant reactivity impairment for the immunodominant antigenic bands (57, 43 and 35 kDa) recognized by sera of vaccinated heifers, reflecting in lower vaccinal antibody avidity for such antigenic markers. In conclusion, iELISAs with B. abortus S-LPS using Protein A/peroxidase showed to be a potential tool for discriminating S19 vaccinal responses from B. abortus infection due to a preferential detection of IgG2 subclasses. Also, B. abortus-specific IgG reactivity profile in avidity-immunoblot demonstrated that vaccinal antibodies showed a lower avidity for antigenic components with apparent molecular masses of 57, 43 and 35 kDa, thus representing potential antigenic markers to differentiate vaccinated from naturally infected cattle.