Tsb st

A virulent Bb strain of canine origin was used for challenge. This challenge strain was grown in TSB-ST (BioMérieux SA, Marcy l’étoile, France) at 37 °C, with shaking at 180 rpm, for 6 h. The bacterial culture was collected when an OD₆₀₀ of 1 was reached (approximately equivalent to 1 × 10⁹ colony-forming units (CFU)/mL). On D35, all the study animals were anesthetized with isoflurane. Rats were inoculated with 25 µL of the challenge strain in each nostril, resulting in a total dose of 50 µL of the strain in PBS, or a titer of 1 × 10³ CFU/animal. The diluted bacterial cultures were back-titrated to confirm the titers used (CFU/animal). Each suspension was serially diluted and 10 µL drops were dispensed onto Bordet Gengou agar containing 15% sheep blood (Becton Dickinson, Heidelberg, Germany).

The live attenuated strain of Bb present in the oral Recombitek vaccine [17 (link)] was used for vaccination. The vaccine strain was grown in TSB-ST (BioMérieux SA, Marcy l’étoile, France) at 37 °C, with shaking at 180 rpm, for 14 h. The bacterium was cultured until an OD₆₀₀ of 2.5 (approximate equivalent to 1.5 × 10⁹ CFU/mL) was reached. At D0 and D21, female Wistar rats (3–4 weeks old) were anesthetized with isoflurane for nasal administration. The live attenuated vaccine strain was then administered intranasally (10⁶ CFU/animal) or orally (10⁸ CFU/animal) with a micropipette. The bacterium was diluted in phosphate-buffered saline (PBS); the inoculation volume used was 50 µL/animal for intranasal administration (25 µL in each nostril), and 100 µL for oral micropipette-guided drug administration (MDA) [26 (link)]. One group of animals received PBS under the same conditions, as a non-vaccinated control.