Inactivated Mycobacterium bovis Vaccine Protocol

The M. bovis strain used was a first passage level culture isolated from a naturally infected wild boar in Coletsos medium. The isolate was propagated in Middlebrook 7H9 broth enriched with OADC for 2–3 weeks. Cells were harvested by centrifugation at 2500 x g for 20 minutes and washed twice in PBS. The bacterial pellet was re-suspended in PBS and declumped using a fine needle syringe. The turbidity of this suspension was adjusted to an optical density of 1 McFarland unit. Before inactivation, tenfold serial dilutions were prepared and plated in agar-solidified 7H9 with OADC in quadruplicate to assess the number of cfu in the inoculum. The inoculum was then inactivated in a water bath at 80°C for 30 minutes. Animals in “parenteral inactivated vaccine” and “oral inactivated vaccine” groups were administered with approximately 6×10⁶ bacteria according to cfu counts. The parenteral vaccine (1 ml) was prepared using Montanide ISA 50 V, an oily adjuvant of mannide oleate and mineral oil (Seppic, Castres, France). The oral vaccine consisted of 2 ml of PBS containing the inactivated mycobacteria. This inactivated vaccine was again cultured in duplicate to assure absence of viable M. bovis.