Comparison of subcutaneous and conjunctival Brucella melitensis Rev.1 vaccination in small ruminants: Serological response and post-challenge bacterial dissemination

Brucella melitensis Rev.1 is widely used to control brucellosis in sheep and goats, but the route of administration may affect both protection and post-vaccination serological interference. This study compared subcutaneous (SC) and conjunctival (VC) administration of the live attenuated B. melitensis Rev.1 vaccine in small ruminants. Twenty-five local-breed sheep and goats were assigned to subcutaneous vaccination (SC, n = 10), conjunctival vaccination (VC, n = 10) or a non-vaccinated challenged control group (n = 5). The vaccinated groups differed simultaneously in route, dose and excipient (SC: 2 × 10 9 CFU in 2 mL of conventional diluent; VC: 1 × 10 9 CFU in 40 µL of diluent containing 5% Montanide Gel-01), so the comparison reflects these three factors combined rather than route alone. Serology was monitored for 15 months by indirect ELISA and the Rose Bengal test, after which all animals were challenged conjunctivally with virulent B. melitensis 16 M and euthanised 15 days later. Subcutaneous vaccination induced a persistent antibody response that remained above baseline at month 15 (S/P% 181.4 ± 49.4), with 4/10 animals still Rose Bengal-positive. Conjunctival vaccination produced a transient response that never reached the positivity threshold (maximum 36.1%), and all VC animals were Rose Bengal-negative from month 6 onward (month 6: 10/10 versus 0/10; Holm-adjusted p = 6.5 × 10 -5 ). After challenge, Brucella was recovered from 8/10 SC, 4/10 VC and 5/5 control animals; the difference between the vaccinated groups was not significant (p = 0.17). Bacterial burden was significantly lower in VC (median 0 versus 262.5 colonies per animal; p = 0.0052), and recovery in VC was confined to the lymph nodes, whereas SC animals also yielded Brucella from spleen and liver. All isolates were identified as strain 16 M. Neither route conferred sterilising immunity. Within the limits of a small cohort and a confounded design, the conjunctival formulation combined reduced serological persistence with more restricted dissemination a combination that may be advantageous where vaccination must remain compatible with serological surveillance.