Re-suspension of T144 vaccine cultures of Mycoplasma mycoides subsp. mycoides SC in 1 molar MgSO4 causes a drop in pH and a rapid reduction in titre

The effect of reconstituting freeze-dried vaccine cultures of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) strain T144 grown in standard vaccine medium using variable quantities of un-buffered solutions of 1 M MgSO4 (the current O.I.E.-recommended procedure) has been investigated. Compared to the culture pH prior to desiccation, a drop of up to 2.2 pH units was observed, dependent upon the volume and pH of 1 M MgSO4 used (1–30× original culture volume, using 1 M MgSO4 in the pH range 5.4–7.6). This pH drop appears to be due to the removal of the HPO42− buffer capacity of the medium by the formation of insoluble Mg3(PO4)2 and the release of free H+ ions. As a result of this lower pH, markedly reduced culture viability was observed over an 8-h period at 37°C for vaccines re-suspended in 1 M MgSO4 (ca. 6 log10 drop in titre) compared to re-suspension in dH2O (ca. 1.5 log10 drop in titre). Re-suspension in 1 M MgSO4 did exhibit a thermoprotective effect at 46°C, but only when the pH was maintained above pH 7.0 by use of HEPES-buffered growth medium (1 log10 drop in titre compared to 6 log10 drop using dH2O over an 8-h period). Since all current O.I.E.-recommended growth media for MmmSC are based upon a phosphate buffer system, it is therefore recommended that the use of 1 M MgSO4 as a reconstitution fluid be discontinued as soon as possible and buffered saline be used instead. The use of this reconstitution procedure with the T144 vaccine strain could be a significant factor in the poor efficacy observed with current freeze-dried vaccines against contagious bovine pleuropneumonia in Africa.