We developed a guinea pig model of extrapulmonary dissemination and utilized it to study CNS dissemination of three different laboratory strains of M. tuberculosis after an aerosol infection. Although all three strains evaluated in this study grew exponentially in the lungs and had similar bacterial burdens at the time of extrapulmonary dissemination (day 14), M. tuberculosis CDC1551 disseminated to the CNS significantly more than the two H37Rv strains. Brain CFU counts were higher than CFU in the whole blood volume of the animal at every time point after day 14, precluding the possibility of brain tissue contamination by extraneous bacteria from the blood. This increased dissemination to the CNS may be explained partially by the higher bacterial burdens noted in the spleens of guinea pigs infected with M. tuberculosis CDC1551 at day 14, although animals infected with H37Rv TAMU had comparable bacterial burdens but significantly lower dissemination to the CNS. Moreover, CNS invasion by M. tuberculosis H37Rv strains continued to be significantly lower than CDC1551 at later time points (days 28 and 56), while spleen CFUs were not different amongst the 3 strains.
Kaplan and colleagues have examined the virulence of M. tuberculosis
strains in a rabbit model of TB meningitis. In contrast to our results, they demonstrated that rabbits infected with the M. tuberculosis
H37Rv strain had higher bacterial burdens in the cerebrospinal fluid (CSF) and brain, with increased dissemination to other organs compared with those infected with CDC15513
. However, these studies utilized direct intracisternal inoculation of bacteria into the CSF and evaluated subsequent dissemination from the CNS to the lungs or liver. This model therefore does not reflect CNS TB as it occurs in humans, where bacteria disseminate to the CNS via blood. Palanisamy et al
. have also examined extrapulmonary dissemination of selected M. tuberculosis
strains in the guinea pig aerosol model. Although dissemination to the CNS was not included in their studies, M. tuberculosis
CDC1551 strain was found to be more virulent than the H37Rv strain7
. Further, dissemination to the spleen was no different between the CDC1551 and H37Rv strains (evaluated 30 days after infection), which is consistent with our data.
As an increasing number of clinically-derived strains are genotyped, it is becoming apparent that the profile of TB disease is likely to be influenced by the infecting strain. Multiple reports have shown the association of different M. tuberculosis
strains with extrapulmonary dissemination. Garcia de Viedma et al.
have shown that TB patients may be concurrently infected with distinct M. tuberculosis
strains that inhabit pulmonary and extra-pulmonary sites8, 9
. A recent study by Hesseling et al.
demonstrates that children infected with the M. tuberculosis
Beijing or S genotypes were more likely to have extra-pulmonary TB compared with children infected with the LAM genotype10
. Similarly, a report by Caws et al.
demonstrates that the Euro-American lineage of M. tuberculosis
is less capable of CNS dissemination than other M. tuberculosis
. Another recent report, utilizing an experimental mouse model, demonstrates that clinical strains isolated from patients with TB meningitis, but not pulmonary TB, disseminate extensively to the CNS12
. These data highlight the association of specific M. tuberculosis
strains with extrapulmonary dissemination and CNS TB, and suggest that M. tuberculosis
may have virulence factors that promote CNS dissemination, distinct from those required for pulmonary TB. This hypothesis is consistent with our prior findings in a murine model, suggesting that distinct M. tuberculosis
genes promote CNS dissemination which are not required for survival in lung tissue13
. It should be noted that while we have previously shown that animal passaging of M. tuberculosis
does not correlate with virulence in vivo5, 14
, M. tuberculosis
H37Rv JHU used in this study was extensively passaged in animals. We do not currently have access to genome data for M. tuberculosis
strains H37Rv JHU and TAMU, but this and other studies will be the focus of our future work to better understand and identify the microbial determinants of CNS invasion.
Seeding of bacilli in the CNS did not yield a robust inflammatory response. Moreover, the levels of inflammatory cytokines were far below what is typically observed in the lungs at a similar stage of infection6
. General extrapulmonary dissemination is first observed at day 14, which may account for the spike in an inflammatory response in the CNS at this time point, which subsided over the following two weeks. The increase at the final day 56 time point is likely due to more extensive bacillary replication. It should be noted that mouse TNF-α demonstrates > 92% homology with guinea pig protein, and is thus likely to be cross-reactive. However, the mouse TNF-α ELISA used in this study has not been validated in guinea pigs, and represents a limitation of our data. Observed levels of TNF-α may not, therefore, be linearly quantitative, but instead indicate a qualitative increase in the presence of this inflammatory mediator. Future studies in this model will be further informed by cytokine profiling via RT-PCR analysis15
The limited immune response observed is not surprising, as the CNS displays selective and modified immunity1
and are consistent with our prior studies in the murine model of CNS TB13
. The stage of infection observed in this model represents the intial seeding of bacilli in the meninges and parenchyma. At this early phase antigen presentation and lymphocytic immune surveillance is highly limited in the brain parenchyma16, 17
. As was observed in the seminal studies by Arnold Rich in guinea pigs and rabbits, small foci of bacteria can exist for long periods of time without the onset of inflammatory disease18
. Studies have shown that there is an absence of T-cell responses to PPD testing following intracranial injection of heat-killed Bacillus Calmette-Guérin (BCG) in rats, indicating that BCG escapes immune recognition within the parenchyma19
. Studies have also shown that subsequent peripheral sensitization of the immune system results in the development of an immune-mediated delayed-type hypersensitivity (DTH) response and inflammatory lesions surrounding the heat killed BCG within the CNS20
. These experimental data are in concordance with the clinical observation of delayed “paradoxical” intracranial tuberculomas, which develop in patients several weeks to months following anti-TB therapy21
. Further, it should be noted that though the brain parenchyma has limited immune surveillance, the CSF has a much more robust immune response to foreign antigens16
. This is consistent with the observation that rupture of the “Rich foci” into the CSF-containing subarachnoid space causes diffuse, inflammatory meningitis. We therefore believe the animals in this study to represent a pre-meningitic state. As such, the model is primarily one of dissemination and invasion, and not immune activation.
The guinea pig model of CNS infection described in this study represents an important tool for the future study of dissemination and CNS invasion. The guinea pig provides a number of physiological advantages over the mouse model of CNS dissemination, including higher whole brain bacillary burden, more temporally reliable dissemination events, and an observable, albeit modest, inflammatory response13
. Further use of this model will be useful in distinguishing the capacity of clinical strains to cause CNS disease and in the identification of molecular determinants of bacterial invasion.
In summary, we have demonstrated strain-dependent CNS dissemination of M. tuberculosis in guinea pigs. These data suggest that M. tuberculosis may have virulence factors that promote CNS dissemination, distinct from those required for pulmonary TB. Future studies focusing on the identification of microbial virulence factors that promote CNS dissemination would therefore be essential to a better understanding of the pathogenesis of CNS TB.