Susceptibility of brushtail possums (Trichosurus vulpecula) infected with Mycobacterium bovis is associated with a transient macrophage activation profile

Summary

The Australian brushtail possums are highly susceptible to Mycobacterium bovis and are the principal wildlife reservoir of M. bovis in New Zealand. To better understand the disease process in these animals, brushtail possums were infected by the aerosol route with a virulent strain of M. bovis, and immune parameters measured. M. bovis replicated actively in the lungs of infected animals. Animals began developing macroscopic lung lesions at 4 and 5 weeks following infection, with some lesions appearing in the livers and spleens. Infection determined the emergence of blood lymphocytes which proliferated in response to bovine purified protein derivative from M. bovis (PPD-b) at 3, 4 and 5 weeks. The response to a mitogen (Concanavalin A) waned progressively with time. Infection was associated with a modest increase in the numbers of free lung cells. Nitrite was detectable in the lavage fluids of infected animals at 3 weeks postinfection, but not at 4 and 5 weeks. Macrophage activation in the lungs was evident as alveolar macrophages produced more oxidants, significant levels of nitric oxide (NO), as well as tumor necrosis factor alpha (TNF-$\alpha$) bioactivity at 3 weeks postinfection. However, macrophages from infected animals lost the ability to generate nitrite- and TNF-$\alpha$ generation was depressed at 4 and 5 weeks postinfection, the time at which macroscopic lesions in the lungs became apparent. Alveolar macrophages from animals at 3 weeks postinfection blocked the replication of M. bovis in part via a NO-dependent mechanism, and were more refractory for M. bovis growth than cells from naïve animals to bacterial replication. Alveolar macrophages from animals at 4 and 5 weeks postinfection allowed substantial replication of M. bovis, and no NO-dependent bacteriostatic activity was apparent. Introduction of autologous lymphocytes from the blood of infected animals in co-cultures rendered infected macrophages more resistant to M. bovis replication. We conclude that M. bovis infection in brushtail possums is associated with a transient activation of alveolar macrophages, although in vitro exposure to sensitized T cells can enhance this profile.

Introduction

The Australian brushtail possum (Trichosurus vulpecula) has become a serious pest in New Zealand, in terms of the ecological damage they cause, and also as important reservoirs of Mycobacterium bovis.¹ Indeed, attempts to eradicate bovine tuberculosis from cattle in New Zealand failed due to the existence of this wildlife reservoir of infection.² Experimental models of infection in these animals suggests that they are remarkably susceptible to infections with M. bovis.3, 4 Solid evidence suggests that management of M. bovis infections in cattle will require strategies for the control of M. bovis infections in species that act as reservoirs, such as the brushtail possums in New Zealand.²

The sequence of immune events in mice infected with Mycobacterium tuberculosis, and to a lesser extent in humans, has been studied in considerable detail, and has allowed the delineation of some key factors (cells, cytokines) involved in determining resistance towards that pathogen. Infection with M. tuberculosis in mice determines the emergence of CD4⁺-T cells that release interferon gamma (IFN-$\gamma$).⁵ In turn, this cytokine activates macrophages to curtail bacterial replication via the induction of nitric oxide (NO) release which inactivates the bacteria.⁶ Similarly, humans deficient in the IFN-$\gamma$ pathway are more susceptible to mycobacterial infections, suggesting the relevance of that pathway of resistance in humans.⁷ The definition of such immune pathways in other species, including the brushtail possums, has not been forthcoming. Possums infected with M. bovis develop vigorous peripheral blood lymphocyte proliferative responses to purified protein derivative from M. bovis (PPD-b), and vaccination with Bacille Calmette-Guerin (BCG) renders these animals more resistant to subsequent challenge.⁸ A better understanding of immune correlates of protection in M. bovis-infected possums would be desirable if progress towards rational vaccinating strategies for these animals is to be achieved. However, the mechanisms involved in the induction of protective immunity conferred by BCG have not been studied in great detail. Recent preliminary studies have shown that possum macrophages are permissive hosts for the replication of virulent mycobacteria in vitro.⁹ Collectively, these data suggest that immunity to tuberculosis in brushtail possums can be studied via methodologies that have been developed to monitor the development of immunity to tuberculosis in mice or humans.

We report on a series of immune events that follow the aerosol infection of brushtail possums with M. bovis which help to explain the high susceptibility of possums to M. bovis infection.