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Mycobacteria are likely to encounter acidic pH in the environments they inhabit ; however intracellular pH homeostasis has not been investigated in these bacteria. In this study, Mycobacterium smegmatis and Mycobacterium bovis [Bacille Calmette-Gue! rin (BCG)] were used as examples of fast-and slowgrowing mycobacteria, respectively, to study biochemical and physiological responses to acidic pH. M. smegmatis and M. bovis BCG were able to grow at pH values of 45 and 50, respectively, suggesting the ability to regulate internal pH. Both species of mycobacteria maintained their internal pH between pH 61 and 72 when exposed to decreasing external pH and the maximum ∆pH observed was approximately 21 to 23 units for both bacteria. The ∆pH of M. smegmatis at external pH 50 was dissipated by protonophores (e.g. carbonyl cyanide m-chlorophenylhydrazone), ionophores (e.g. monensin and nigericin) and N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocating F 1 F 0 -ATPase. These results demonstrate that permeability of the cytoplasmic membrane to protons and proton extrusion by the F 1 F 0 -ATPase plays a key role in maintaining internal pH near neutral. Correlations between measured internal pH and cell viability indicated that the lethal internal pH for both strains of mycobacteria was less than pH 60. Compounds that decreased internal pH caused a rapid decrease in cell survival at acidic pH, but not at neutral pH. These data indicate that both strains of mycobacteria exhibit intracellular pH homeostasis and this was crucial for the survival of these bacteria at acidic pH values.

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Bovine tuberculosis (Tb) caused by Mycobacterium bovis has proved refractory to eradication from domestic livestock in countries with wildlife disease reservoirs. Vaccination of wild hosts offers a way of controlling Tb in livestock without wildlife culling. This study was conducted in a Tb-endemic region of New Zealand, where the introduced Australian brushtail possum (Trichosurus vulpecula) is the main wildlife reservoir of Tb. Possums were trapped and vaccinated using a prototype oraldelivery system to deliver the Tb vaccine bacille Calmette -Guerin. Vaccinated and control possums were matched according to age, sex and location, re-trapped bimonthly and assessed for Tb status by palpation and lesion aspiration; the site was depopulated after 2 years and post-mortem examinations were conducted to further identify clinical Tb cases and subclinical infection. Significantly fewer culture-confirmed Tb cases were recorded in vaccinated possums (1/51) compared with control animals (12/71); the transition probability from susceptible to infected was significantly reduced in both males and females by vaccination. Vaccine efficacy was estimated at 95 per cent (87-100%) for females and 96 per cent (82-99%) for males. Hence, this trial demonstrates that orally delivered live bacterial vaccines can significantly protect wildlife against natural disease exposure, indicating that wildlife vaccination, along with existing control methods, could be used to eradicate Tb from domestic animals.

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We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Twenty-two white-tailed deer were divided into four groups. One group (n=5) received 10(9) colony-forming units (cfu) BCG via a lipid-formulated oral bait; one group (n=5) received 10(9) cfu BCG in culture directly to the oropharynx, one group (n=6) was vaccinated with 10(6) cfu BCG subcutaneously, and one group served as a control and received culture media directly to the oropharynx (n=6). All animals were challenged 3 mo after vaccination. Five months postchallenge the animals were examined for lesions. Results indicate that both oral forms of BCG and parenterally administered BCG offered significant protection against M. bovis challenge as compared to controls. This study suggests that oral BCG vaccination may be a feasible means of controlling bovine tuberculosis in wild white-tailed deer populations.

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