Identification of Leukotoxin and other vaccine candidate proteins in a Mannheimia haemolytica commercial antigen

Tucci, Paula; Estevez, Verónica; Becco, Lorena; Cabrera-Cabrera, Florencia; Grotiuz, Germán; Reolon, Eduardo; Marín, Mónica

2.1. Laboratory scale M. haemolytica culture A seed culture was prepared by inoculation of an aliquot of master seed bank (MSB) of M. haemolytica serotype A1 (production strain code B059) in TSB-BHI (Tryptic Soy Broth, Brain Heart Infusion, Porcine) and incubation at 37.0 ± 1.0°C, for 12 h with orbital agitation (220 rpm). Inoculum was prepared by a 1:100 dilution of the seed culture in TSB-BHI and further incubation at 37.0 ± 1.0°C with orbital agitation until an optical density at 600 nm (OD 600nm ) of 0.2 to 0.3 units was reached. A benchtop fermentor (Biostat A, New Brunswick) with 2 L of fermentation media (TSB-BHI supplemented with 0.4% glucose) was inoculated with 20 mL of the inoculum culture (1:100 dilution). The fermentation culture was grown at 37.0 ± 1.0°C with dissolved oxygen set above 40% and variable Article No~e00158 agitation. During fermentation, samples were taken every one hour to measure OD 600nm , dissolved oxygen (%), glucose concentration (mg/dL) and pH. The culture was harvested when it reached stationary growth phase. Samples and the final culture were centrifuged at 8000×g for 30 min at 4°C and culture supernatants were stored at −20°C.