Research Article: The Fischer 344 Rat Reflects Human Susceptibility to Francisella Pulmonary Challenge and Provides a New Platform for Virulence and Protection Studies

Date Published: April 1, 2010

Publisher: Public Library of Science

Author(s): Heather J. Ray, Ping Chu, Terry H. Wu, C. Rick Lyons, Ashlesh K. Murthy, M. Neal Guentzel, Karl E. Klose, Bernard P. Arulanandam, Raphael H. Valdivia.

Abstract: The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies.

We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative of subsp. holarctica) and subsp. novicida. Within BMDM, subsp. tularensis and LVS showed minimal replication, subsp. novicida showed marginal replication, and subsp. holartica replicated robustly. The limited intramacrophage replication of subsp. tularensis and novicida strains was correlated with the induction of nitric oxide production. Importantly, Fischer 344 rats that survived pulmonary infection with subsp. novicida were markedly protected against subsequent pulmonary challenge with subsp. tularensis, suggesting that subsp. novicida may be a useful platform for the development of vaccines against subsp. tularensis.

The Fischer 344 rat exhibits similar sensitivity to F. tularensis strains as that reported for humans, and thus the Fischer 344 ray may serve as a better animal model for tularemia vaccine development.

Partial Text: Francisella tularensis is a gram negative bacterium that is the causative agent of the human disease tularemia [1], [2]. There is currently no licensed vaccine for human use, but extensive efforts are underway to identify potential vaccine candidates for this biothreat agent. Most of our current understanding of pathogenesis and host immune responses comes from studies using the mouse model of F. tularensis infection [3], [4], [5], [6], [7]. While the murine tularemia model has extended our understanding of the disease process, the extreme sensitivity of the mouse to F. tularensis challenge limits the usefulness of this model in protection studies.

Macrophages are important host cells for intracellular F. tularensis replication [4], [7], [27]. In mouse BMDM, the type A, type B, LVS and subsp. novicida strains replicate to 3–4 log10 within 24 h of challenge [7], [13], [34]. In contrast, while subsp. novicida is considered avirulent in humans, the bacterium was found to replicate 3–4 log10 by 72 h of culture in human peripheral blood monocytes and monocyte derived macrophages (hMDM) [33]. LVS and subsp. tularensis also have been shown to replicate in hMDMs, however opsonization with autologous serum is required for efficient uptake and replication of these two strains [26], [27], [35]. In the rat, we found that LVS was unable to replicate in Fischer 344 BMDM, and that while subsp. novicida was phagocytosed to a greater extent initially, replication was limited to 1–2 log10 by 24 h. Subsp. tularensis and holarctica were taken up in relatively low numbers initially, followed by minimal replication with subsp. tularensis, but continued replication (2–3 log10) with subsp. holarctica. Therefore, while absolute numbers of subsp. novicida and subsp. holarctica recovered were similar (approx. 105 CFU), subsp. holarctica was able to replicate to a much greater degree. These differences in intramacrophage replication may not be attributed to a defect in uptake, since Fischer 344 rat BMDM exhibited better basal phagocytic ability to mouse BMDM. Moreover, inclusion of homologous rat serum did not facilitate increased uptake or replication of LVS or subsp. tularensis in rat BMDM, in contrast to reports with human macrophages. One of the primary differences between human and rat macrophage is the capacity to produce NO in vitro[36], [37]. Whereas human macrophages produce minimal NO upon LPS stimulation, rat macrophages have been shown to express 5-fold greater production of NO than mouse cells [38]. To this end, we found that subsp. novicida and subsp. tularensis induced the production of NO, and that addition of a NO inhibitor during the culture period increased the overall replication of both strains by 3 log10 at 72 h. This suggests that Fischer 344 rat macrophages are capable of containing the replication of these subsp. at least in part by the induction of NO. Thus, suppression of NO production resulted in a bacterial replication profile which more closely resembled that of hMDMs. The in vitro intracellular replication profile of the Francisella strains tested corresponded to in vivo susceptibility of Fischer 344 rats to pulmonary challenge. Importantly, Fischer 344 rats were markedly resistant to both LVS and subsp. novicida, while mice are highly sensitive to these two avirulent human strains [11], [13]. While both subsp. tularensis and holarctica causes serious disease in humans, the former is associated with much higher risk of mortality, which also is reflected in the rat model by the lower pulmonary LD50 dose of subsp. tularensis. Although there was minimal replication of subsp. tularensis within BMDM, there was clear evidence for robust bacterial replication in primary hepatocytes from the liver, a known site for disseminated bacterial replication [10], [24]. However, this does not rule out the ability of subsp. tularensis to replicate in primary macrophages isolated from the lung (alveolar macrophages) or peritoneal cavity (ongoing studies).