Diagnostic Performance of Rose Bengal, Competitive ELISA, and Native Hapten (NH) Assays in S19 Mass‐Vaccinated Cattle in an Endemic Brucellosis Environment

Controlling bovine brucellosis requires reliable diagnostics and effective vaccination strategies. We evaluated the performance of three serological assays—the rose bengal test (RBT), competitive ELISA (cELISA), and agar gel immunodiffusion (AGID) with native hapten (NH)+lipopolysaccharide (LPS) antigen—following Brucella abortus S19 vaccination under different protocols. Serum samples were collected from two cattle populations in Costa Rica: (i) a brucellosis‐free cohort of 25 heifers (Farm 1) vaccinated once with a reduced dose of S19 (5 × 10 9 CFU) via either the conjunctival ( n = 5) or subcutaneous ( n = 20) route, and (ii) a previously vaccinated herd with B. abortus RB51 (Farm 2) of 253 cattle, cleared of Brucella infection by test and slaughter, was then vaccinated with S19 (either 5 × 10 10 or 5 × 10 9 CFU, conjunctival or subcutaneous). All sera were tested by RBT, cELISA, and AGID using standard procedures. In validation with control sera, RBT, cELISA, and AGID NH+LPS each correctly identified all 35 positive and all 35 negative sera, while AGID NH+LPS correctly identified 33 of 35 positive sera and all negative sera. In Farm 1, conjunctival vaccination induced only a transient antibody response: all heifers were seronegative by 7 weeks postvaccination, and none developed NH‐specific precipitin lines. Subcutaneous vaccination produced stronger and longer‐lasting responses: ~20% of animals remained RBT/cELISA‐positive at 1 year (5% at 2 years), though none reacted to NH. In Farm 2, S19 vaccination elicited sustained RBT and cELISA seropositivity, especially after subcutaneous administration. About 5% of animals developed NH‐specific precipitin lines over time, consistent with an anamnestic response to residual infection. These findings show that vaccination route affects serological outcomes: conjunctival S19 vaccination yields minimal long‐term interference, while subcutaneous vaccination prolongs seropositivity. The AGID NH+LPS test helped identify actively infected animals in vaccinated herds. We recommend S19 mass vaccination via the conjunctival route and caution against using cELISA as a routine confirmatory test following RBT in endemic settings.