Susceptibility of immunodeficient mice to aerosol and systemic infection with virulent strains of Francisella tularensis

Microbial Pathogenesis 36 (2004) 311–318 www.elsevier.com/locate/micpath

Susceptibility of immunodeficient mice to aerosol and systemic infection with virulent strains of Francisella tularensis Wangxue Chen*, Rhonda KuoLee, Hua Shen, J. Wayne Conlan Institute for Biological Sciences, National Research Council Canada, Ottawa, Ont., Canada K1A 0R6 Received 6 November 2003; received in revised form 3 February 2004; accepted 3 February 2004

Abstract Previous studies have shown that IFN-g, TNF-a and NOS-2, but not B cells, are crucial for host defense against primary systemic infection with the attenuated live vaccine strain (LVS) of Francisella tularensis. In this study, we examined the importance of these and additional immune components in host resistance against infection with virulent strains of F. tularensis initiated by systemic and airborne routes. Wildtype (WT) mice and mice deficient in IFN-g, TNFR1R2, NOS-2, or B cells were equally susceptible to low dose (,10 colony forming units) aerosol or intradermal challenge with virulent type B F. tularensis, and succumbed to the infection between days 6 and 8 post-inoculation. Quantitative bacteriology showed that IFN-g 2 /2 and B cell 2 /2 mice consistently harbored up to one log10 more bacteria in their lungs, spleens and livers than WT mice at day 5 post aerosol exposure. Surprisingly, however, compared to other strains of KO mice and WT control mice, IFN-g 2 /2 mice showed only mild liver damage as assessed by histopathology and liver function tests. Additional experiments established that even mice with broad immunodeficiency (SCID, neutropenic, splenectomized or thymectomized mice and mice treated with corticosteroid) were no more susceptible to aerosol-initiated infection with virulent type B or type A F. tularensis than immunosufficient control mice. Combined, our results indicate that, unlike LVS, normal type A and type B F. tularensis strains are so extremely virulent that even immunocompetent mice are virtually defenseless to low dose aerosol and intradermal challenges with them. Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. Keywords: Francisella tularensis; Virulent; Immunodeficient; Pathogenesis; Mouse model

1. Introduction Francisella tularensis is a facultative intracellular bacterial pathogen capable of causing disease, tularemia, in many mammalian species including humans [1]. Naturally, it can initiate human infection through arthropod bites, direct contact with infected tissues, inhalation, or ingestion [1]. Two subspecies of F. tularensis, F. tularensis subspecies tularensis (type A) and F. tularensis subspecies holarctica (type B), exist and both are highly infectious for humans [1,2]. However, only type A strains of F. tularensis routinely cause lethal infection in people especially following exposure to infectious aerosols of the pathogen [1]; inhalation of as few as 10 virulent type A bacilli is sufficient to initiate severe disease [3]. Although type B F. tularensis is less virulent than type A for higher mammals and nonfatal for humans, it is more widely distributed * Corresponding author. Tel.: þ 1-613-991-0924; fax: þ1-613-952-9092. E-mail address: [email protected] (W. Chen).

throughout the Northern hemisphere and is the cause of most clinical tularemia. Because of its high level of infectivity and its ability to be disseminated as an aerosol, F. tularensis has been classified as a Category A biological warfare agent by The Working Group on Civilian Biodefense [4]. Currently, relatively little is known about the pathogenesis of and immunity to virulent F. tularensis infection. To date most of our knowledge on immunopathogenesis of F. tularensis infection has been derived from studies of systemic infection via unnatural routes with the attenuated live vaccine strain (LVS) of F. tularensis. LVS was derived from a virulent type B strain of F. tularensis by passage on culture media [5]. LVS is attenuated for humans, but remains virulent for mice, especially when administered through unnatural i.v and i.p. routes [6]. With such mouse models, it has been demonstrated that T cells, neutrophils, TNF-a, and IFN-g play crucial roles in controlling primary systemic infections with LVS [7]. Furthermore, the role of neutrophils and IFN-g in host defense against primary LVS

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infection appears to vary with the route of infection [8]. On the other hand, B cells play only a marginal role in resistance to primary LVS infection [9]. However, there are genuine concerns about using LVS as a surrogate for more virulent strains of the pathogen in infection and immunity studies. For instance, the latter strains have far lower LD50 than LVS when administered by natural routes [10,11], induce different pathologies [11], and are far less susceptible to antibody-mediated killing than LVS [12]. Thus, studies using experimental animals infected by natural routes with virulent F. tularensis strains will be necessary for further understanding of the pathogenesis of and immunity to human tularemia, and for the development of effective prevention and treatment strategies. We have recently established mouse models of low-dose aerosol and intradermal infection with virulent type A and type B strains of F. tularensis, which mimic many clinical and pathological aspects of untreated human tularemia [11]. The objective of this study was to use these models to determine whether or not hosts with defined or broad immunodeficiency are more susceptible to infection with virulent F. tularensis than immunosufficient mice. The results presented here show that, unlike the situation with LVS, mice deficient in IFN-g, TNF receptors 1 and 2, NOS2 or B cells were no more susceptible to aerosol or intradermal infection with virulent type B F. tularensis than wild-type (WT) mice. However, IFN-g 2 /2 mice showed a higher tissue bacterial burden and milder liver damage than wild-type mice. Furthermore, mice with broad immunodeficiency (SCID, neutropenic, splenectomized, thymectomized or corticosteroid-treated mice) were no more susceptible than WT mice to aerosol infection with virulent type A and type B F. tularensis.

2. Results and discussion 2.1. Susceptibility to aerosol or intradermal infection with attenuated and virulent type B F. tularensis of mice deficient in IFN-g, TNFR1R2, NOS-2 or B cells Previously published studies have shown that the cytokines TNF-a and IFN-g, and the enzyme NOS-2, but not B cells, play crucial roles in host defense against primary systemic LVS infection [7]. In this study, we undertook a series of experiments to examine whether or not these and other immune components also play an important role in host defense against virulent strains of F. tularensis. We began by determining the susceptibility of mice deficient in IFN-g, TNFR1R2, NOS-2 or B cells to aerosol or intradermal challenge with LVS or a virulent type B strain (strain 108) of F. tularensis (Table 1). As expected from previously published work [13 –15], IFN-g 2 /2 and TNFR1R2 2 /2 mice were highly susceptible to intradermal challenge with very low doses (, 20 cfu) of LVS in that all such mice succumbed to the infection by days 8 and 10, respectively, whereas WT mice survived i.d. challenge with a 10,000-fold larger inoculum. IFN-g 2 /2 and TNFR1R2 2 /2 mice were also moderately susceptible to aerosol-initiated infection with 10 2 cfu LVS with a mortality of 60 – 80%, compared to 0% for similarly challenged WT mice. In contrast, B-cell 2 /2 and NOS2 2 /2 mice were much more resistant than IFN-g 2 /2 and TNFR1R2 2 /2 mice to low dose i.d. or aerosol challenge with LVS. The fact that some of these KO mice survived could simply reflect the fact that they failed to retain any LVS in their lungs during the aerosol exposure. However, historically this has not been a problem with the challenge dose employed compared to the 10 fold lower

Table 1 Comparison of the survival rates of different strains of KO mice to aerosol or intradermal infection with virulent (strain 108) and attenuated (LVS) type B F. tularensis Mouse strain

Survival of mice challenged by aerosol or i.d. routes with LVS, MTDa (range; % survival) Aerosol

IFN-g 2/2 B-cell 2/2 NOS-2 2/2 TNFR1R2 2/2 C57BL/6

15 (10–21; 40%) $21c (.21; 100%) $21 (.21; 100%) 12 (11-21; 20%) $21 (.21; 100%)

#108, MTD (range; % survival) i.d.

Aerosol

i.d.

8 (7– 8; 0%) $21 ($21; 100%) $21 ($21; 100%) 10 (8-10; 0%) $21($21; 100%)

7 7 7 7 7

6 (6–15b; 25%) 6.5 (6–7; 0%) 6.5 (6–7; 0%) 7 (7–8; 0%) 7 (6–8; 0%)

(7– 7; 0%) (7– 8; 0%) (6– 7; 0%) (7– 7; 0%) (6– 7; 0%)

Different strains of KO mice ðn ¼ 4 – 5Þ were challenged with a low dose aerosol (,10 cfu) or i.d. (,20 cfu) inoculum of virulent type B F. tularensis or LVS (102 cfu aerosol and ,20 cfu i.d.) and their survival was monitored daily. WT mice (C57BL/6) were challenged with similar doses of the respective pathogens, except the control mice for i.d. challenges with LVS that received 2 £ 105 cfu. a MTD: median time to death (days). b Experiment was terminated on the day indicated. No F. tularensis was cultured and no pathological changes were observed in the spleen from the surviving mouse, indicating that the mouse was not infected during the i.d. exposure. c The LVS experiment was terminated on day 21 of infection, and no LVS was cultured from the spleen of the surviving mice.

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dose employed for virulent type A and type B strains of F. tularensis. Instead, the somewhat greater resistance of the TNFR1R2 2 /2 and IFN-g 2 /2 mice to aerosol versus i.d. challenge with LVS reflects a pattern we have previously seen in neutropenic and IFN-g-depleted mice [8] that we have interpreted as evidence of the site-specific nature of certain anti-Francisella defenses. Surprisingly, none of the immunodeficient mouse strains examined was more susceptible to low dose (, 10 cfu) aerosol or intradermal challenge with virulent type B F. tularensis, in that all such mice died at the same time as WT mice (Table 1). Given that virulent type A F. tularensis is even more virulent for immunocompetent C57BL/6 mice than virulent type B F. tularensis [11], it seems reasonable to expect a similar result would have been obtained if the former pathogen had been used in these experiments. These findings imply that host defense mechanisms effective against LVS are impotent against virulent F. tularensis. Since IFN-g, TNF-a and NOS-2 are important defenses against most intracellular pathogen infections, our results suggest that wild-type F. tularensis has evolved unique strategies to overcome the immune mechanisms governed by these molecules. Presumably, this accounts for the extreme virulence of wild-type F. tularensis for the infected host. The corollary is that LVS is attenuated because it has lost its ability to resist these host defenses, but can still behave like virulent strains in their absence. 2.2. Bacteriologic and histopathologic studies on immunodeficient mice following aerosol infection with virulent type B F. tularensis Although the above results clearly showed that mice deficient in IFN-g, TNFR1R2, NOS-2 or B cells exhibit a similar susceptibility to virulent type B F. tularensis infection as WT mice, it remained possible that different pathogenic mechanisms were involved in each case. To examine this possibility, groups of mice were infected by the aerosol route with , 10 cfu virulent type B F. tularensis on day 0 and sacrificed on day 5, about 48 h before the expected death of the majority of infected mice, and at a time when the bacterial burden reaches its peak in the lungs, livers and spleens [11]. Their lungs, livers and spleens were collected and used for quantitative bacteriology and histopathology. As can been seen in Fig. 1, there were no statistically significant differences in the bacterial burdens in the lung, liver and spleen among different strains of KO mice and WT mice. However, infected organs from IFNg 2 /2 and B cell 2 /2 mice consistently contained about 0.5 – 1.0 log10 more bacteria than those of WT mice. However, as this represents only 2 – 4 additional doublings for a rapidly proliferating pathogen, these results suggest that the net growth rate of virulent type B F. tularensis in different strains of KO mice is similar to that in WT mice. At necropsy, the lungs from all mice showed no remarkable gross changes, and remained floating in

Fig. 1. Burdens of type B F. tularensis in different strains of KO mice and WT (C57BL/6) mice. Mice were challenged by aerosol with a low dose (,10 cfu) of virulent type B F. tularensis (strain 108). Bacterial burdens in livers, spleens, and lungs were determined at day 5 post-inoculation. The lower and upper limits of bacterial detection were log103.30 and log108.60, respectively. Similar results were obtained when mice were killed on day 4 after aerosol challenge.

the fixative, a situation we have found previously in both BALB/c and C57BL/6 mice [11]. Occasionally, the livers were slightly mottled, yellowish, and had a mildly friable and flaccid texture. The spleens were slightly enlarged in WT mice but not in any strains of KO mice. Histologically, the livers from most mice except those from IFN-g 2 /2 mice showed multifocal inflammatory necrosis of small to medium size throughout the section (Fig. 2B –E). Some adjacent necrotic foci were coalescing to produce large areas of hepatic necrosis. However, the livers from IFNg 2 /2 mice showed surprisingly mild lesions consisting of only occasional hepatocyte necrosis and infiltration with a few inflammatory cells, as compared to other KO mice or WT mice (Fig. 2A). Blood biochemical analysis showed that the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were only moderately elevated in

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Fig. 2. Liver histopathology in different strains of mice killed on day 5 after aerosol infection with ,10 cfu virulent type B F. tularensis. (A) Liver from an IFN-g 2 /2 mouse showing the presence of limited focal accumulation of mixed mononuclear cells and neutrophils. (B-E) Livers from TNFR1R2 2 /2 (B), B cell 2 /2 (C), NOS-2 2 /2 (D) and WT mice (E) showing medium sized inflammatory infiltrations by mixed mononuclear cells and neutrophils and the necrosis of hepatocytes (arrows). Bars ¼ 40 mm.

IFN-g 2 /2 , TNFR1R2 2 /2 and B cell 2 /2 mice compared to NOS-2 2 /2 mice and WT mice (Fig. 3). Although these liver function enzyme results corroborate well with the relatively mild hepatic damage seen in IFN-g 2 /2 mice they were inconsistent with the hepatic histopathology seen in other KO mice and WT mice. The lack of positive correlations between the degree of hepatic necrosis and liver function tests in the present study remains enigmatic, although a similar phenomenon has been observed with a number of other hepatic injuries [16 – 18]. In these studies, it has been postulated that ALT and AST can leak from the cells either through a more permeable hepatocyte membrane [17] or the development of membrane blebs [16] without subsequent cell death. There were no remarkable differences in the extent and severity of histological changes in the lung and spleen

among the different strains of mice. In the lung, there were areas of severe necrosis in which large amounts of necrotic cellular debris, and a few intact alveolar macrophages, and many neutrophils were admixed with varying numbers and sizes of bacterial colonies. The peribronchial and perivascular lymphatics were often moderately to severely dilated and filled with eosinophilic exudates and mixed inflammatory cells. There was mild to moderate pleuritis composed mainly of mononuclear cells. Elsewhere, the alveolar septa were moderately thickened and showed increased numbers of macrophages and other mononuclear cells but seldom neutrophils. Airway epithelial cells were generally not involved although accumulation of inflammatory exudate in the lumens of bronchi and bronchioles could be seen from time to time in the severely affected areas. The spleens from all mice showed moderate to severe loss of lymphoid follicles of the white pulp and multifocal infiltrations of

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Fig. 3. Blood chemistry analysis from different strains of mice ðn ¼ 5Þ challenged by aerosol with a low dose (,10 cfu) of virulent type B F. tularensis. Serum samples were collected at day 5 after aerosol exposure and analyzed for the levels of albumin, blood urea nitrogen, ALT and AST. * p , 0:05 vs. WT mice.

neutrophils admixed with varying numbers of degenerated and necrotic lymphoid cells and sometimes with numerous bacteria. Overall, the histopathological feature was similar to that we’ve described previously for both C57BL/6 and BALB/c mice [11]. 2.3. Effect of neutralization of endogenous IFN-g on the susceptibility of mice to aerosol infection with type B F. tularensis Results from aforementioned experiments showed that, in spite of a similar clinical outcome to other strains of KO mice and WT mice (Table 1), IFN-g 2 /2 mice developed a higher hepatic bacterial burden but a milder liver pathology following low-dose aerosol infection with virulent type B F. tularensis (Figs. 1 and 2). These findings suggest that IFN-g may play a divergent role, though minor in this case, in the development of tularemia following the infection with virulent type B F. tularensis. To examine this possibility and to better define when IFN-g is required during the course of infection, groups of C57BL/6 mice were treated at various time points with Mab R4-6A2 to neutralize the endogenous IFN-g [8]. The mice were then exposed to a low dose aerosol of virulent type B F. tularensis. The clinical progress of these mice was monitored daily. As can be seen in Table 2, neutralization of endogenous IFN-g at the early phase (2 2 and 48 h), late phase (96 h)

or throughout the entire phase (2 2, 48 and 96 h) of the infection had no significant effect on the mortality and median time to death of the infected mice, although the mice treated with anti-IFN-g Mab on day 4 only showed slightly prolonged median time to death and was less sick clinically, thereby verifying that this cytokine by itself does not play a crucial role in the susceptibility of mice to aerosol infection with virulent F. tularensis.

Table 2 The effect of neutralizing endogenous IFN-g on the course of an aerosol challenge with virulent type B F. tularensis R46-A2 treatmenta

Survival (%)

MTDb (range)

– 22, 48, 96 h 48, 96 h 96 h

0 20 20 0

7 (7–8) 7.5 (7–13c) 8 (7–13c) 9 (7–10)

Mice ðn ¼ 5Þ were challenged by nose-only aerosol with ,10 cfu virulent type B F. tularensis and their clinical progress was monitored daily. a Injected by the i.p. route with 0.5 mg IFN-g neutralizing (R46-A2) Mab/mouse in 250 ml PBS or vehicle alone at the indicated times of infection. b MTD: median time to death (days). c Experiment was terminated on day 13 after aerosol exposure. No F. tularensis was cultured and no pathological changes were observed in the spleen from the surviving mouse, indicating that it had not been infected during the aerosol exposure.

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2.4. Susceptibility to aerosol infection with type A and B F. tularensis of mice with broad immunodeficiency Although the results from the studies above have shown that mice with defined single immune component defects are no more susceptible to aerosol or intradermal type B F. tularensis infection, it remained possible that mice with broad immunodeficiency might be. To examine this, SCID, neutropenic, splenectomized, thymectomized and corticosteroid-treated mice were infected by aerosol with low doses (, 10 cfu) of type A or type B F. tularensis, and the clinical progress of the mice was monitored over the course of infection. As shown in Table 3, the susceptibility of the latter immunodeficient mice to aerosol infection with virulent type A and type B F. tularensis was similar to that of control mice in that the majority of mice succumbed to type A F. tularensis infection by day 6 and to type B F. tularensis infection by day 7. In contrast, SCID and neutropenic mice have previously been shown to be highly susceptible to systemic challenge with LVS [15,19]. On the other hand, thymectomized mice were no more susceptible than control mice to systemic LVS infection [20]. In summary, the results from this study have shown several important differences between murine host resistances to virulent strains of F. tularensis versus F. tularensis LVS. In contrast to LVS infections, in which a crucial role of IFN-g and TNF-a for control of the infection has been demonstrated, mice unable to elaborate these cytokines, or with various other immunodeficiencies are no more Table 3 Susceptibility of immunocompromised mice to low dose aerosol challenge with type A and type B F. tularensis Mice

Survival of mice following low dose aerosol challenge with Type A F. tularensis

Control RB6-8C5 treatedb Corticosteroid-treatedd Splenectomized Thymectomized C.B-17 SCID

a

Type B F. tularensis

Survival (%)

MTD (range)

Survival (%)

0 0 0 25 0 0

5 (5–7) 5 (4–5) 5 (5–6) 5 (5–20c) 6 (5–7) 5 (5–6)

0 20 0 0 0 20

MTD (range) 7 (6– 7) 8 (7– 13c) 7 (7– 7) 7 (6– 7) 7 (6– 8) 7.5 (7–13c)

Mice (C57BL/6 except where stated, n ¼ 4 – 5) were exposed to a low dose (,10 cfu) aerosol of virulent type A or type B F. tularensis and their survival was monitored daily. a MTD: median time to death (days). b Neutrophil-depleting Mab RB6-8C5 0.5 mg/mouse i.p. in 250 ml PBS at 22, 48, and 96 h after the infection. c Experiment was terminated on the day indicated. No F. tularensis was cultured or pathological changes were observed in the spleen from the surviving mouse, indicating that the mouse was not infected during the aerosol exposure. d 2.5 mg/mouse, s.c. 4 times, administered weekly, beginning 3 weeks prior to aerosol challenge.

susceptible to aerosol or intradermal infection with virulent strains of F. tularensis. Therefore, caution must be exercised in the future when using studies with LVS infection as a surrogate for infection with more virulent strains of the pathogen, since the former clearly doesn’t always predict the outcome of infection with virulent F. tularensis. However, given that type B F. tularensis is far more virulent for mice than humans, it remains possible that the various host defense mechanisms examined in the present study could operate to control human type B F. tularensis infection. This could be investigated using a more resistant host such as the rabbit or monkey for which type B F. tularensis are infectious, but rarely lethal [21]. However, the range of immunological reagents for studying host defense mechanisms are much more limited for the latter mammalian species. On the other hand, given that type A F. tularensis appears to be as virulent for humans as mice [3], it seems likely that our observations on the futility of the immune defenses of the latter host against this subspecies of the pathogen extend to the human host.

3. Materials and methods 3.1. Mice Female C57BL/6 mice and C.B-17 SCID mice were purchased from Charles Rivers Laboratories (St Constant, Quebec). Female B6.129S7-Ifngtm1 Agt (IFNg 2 /2 ), B6;129S-Tnfrsf1a tm1 Imx Tnfrsf1btm1 Imx tm1 Cgn (TNFR1R2 2 /2 ), B6.129S2-Igh-6 (B cell 2 /2 ) and B6.129P2-NOS2tm1 Lau (NOS2 2 /2 ) mice were purchased from Jackson Laboratory (Bar Harbor, Maine). Mice were housed under specific-pathogen-free conditions in the Animal Facility, Institute for Biological Sciences, National Research Council Canada, and entered into experiments at 8 – 12 weeks of age. Mice were maintained and used in accordance with the recommendations of the Canadian Council on Animal Care Guide to the Care and Use of Experimental Animals. 3.2. Bacteria and mouse inoculation Type A F. tularensis strain FSC33/snMF (strain #33) was originally isolated from a squirrel in Georgia USA [22]. Type B strain FSC108/SBL R45/81 (strain #108) was isolated in Sweden from an ulcer of a tularemia patient [22]. They are maintained in the Francisella Culture Collection of the Swedish Defence Research Agency, Umea, Sweden. They were provided to us by Dr John Cherwonogrodsky (Defence Research Establishment Suffield, Alta., Canada). Stock cultures were grown in modified Mueller Hinton broth, harvested and frozen at 2 70 8C in 2– 4 ml aliquots (1010 cfu/ml) in the presence of 10% (v/v) sucrose. For aerosol exposure, thawed F. tularensis stocks were diluted in Mueller Hinton broth containing 20% (v/v) glycerol; for

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i.d. inoculations stocks were diluted in sterile saline. Actual inoculum’s concentrations were determined by plating 10fold serial dilutions on cysteine heart agar supplemented with 1% (w/v) hemoglobin. Colonies were counted after 48 –72 h of incubation at 37 8C. Intradermal inocula (50 ml/mouse) were injected into the shaved mid-belly. Aerosols of F. tularensis strains were generated with a Lovelace nebulizer operating at a pressure of 40 p.s.i. to produce particles in the 4 –6 mm range required for inhalation and retention in the alveoli [23]. Mice were exposed to these aerosols for 7 min using a commercial nose-only exposure apparatus customized for the current purpose (In-tox Products, Albuquerque, NM), and had an initial retained dose of approximately 10 cfu in the lungs, as described previously [11]. Aerosol exposures were performed in a federally-licensed small animal containment level 3 facility. When performing low-dose aerosol exposures with virulent F. tularensis it is impossible to accurately determine the initial retained dose as repeated experiments by us showed that # 1 cfu could be recovered from homogenized lungs spiked with 10 cfu of the pathogen whereas it is routinely recovered when the inoculating dose is raised to 20 cfu. The former situation has always prevailed in all of our low dose aerosol challenge experiments in which the initial recovered dose has been formally examined, from which we have assumed an initial retained dose of # 10 cfu. Such low doses are required for the studies performed herein given the innate extreme virulence of type A and type B F. tularensis for fully immunocompetent mice to begin with. Unfortunately, when performing low dose aerosol or intradermal challenges an occasional animal fails to receive any bacteria. Such animals are identified in the current study in the experiments in which they occurred.

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examination, the lungs, livers and spleens from five mice of each strain were fixed immediately by immersion in 10% neutral buffered formalin, and processed by standard paraffin embedding methods. Sections were cut 4 mm thick, stained with haematoxylin – eosin (HE) and examined by light microscopy. 3.5. Blood chemistry In some experiments, blood samples were collected from mice sacrificed at day 5 by cardiac puncture. The sera were separated and assayed for the levels of AST, ALT, albumin, and blood urea nitrogen using the Roche Hitachi 917 Analyzer (Vita-Tech, Markham, Ontario). 3.6. Statistical analysis Parametric data were presented as mean ^ standard deviation for each group, and non-parametric data were presented as median (range). Significant differences among groups were analyzed by one-way or two-way ANOVA followed by the Tukey-Kramer multiple comparison test. Differences were considered significant at p , 0:05:

Acknowledgements We thank Tom Devecseri for his expert assistance in the preparation of photomicrography, and Ann Webb and Maria Busa for their technical assistance in the part of this study. This work was supported in part by the National Institutes of Health, USA (AI48474) and by the Institute for Biological Sciences, National Research Council Canada.

3.3. Monoclonal antibody and corticosteroid treatments In some experiments, mice were treated with monoclonal antibody (Mab) RB6-8C5 (0.5 mg/mouse/injection) to deplete them of neutrophils [24] or with Mab R4-6A2 (0.5 mg/mouse/injection) to neutralize endogenous IFN-g [25] by intraperitoneal (i.p.) injection at the indicated times as we described previously [8,19,26]. Broader immunosuppression was elicited by treating mice with hydrocortisone acetate prepared as previously described [27]. This was administered subcutaneously in 2.5 mg doses at the times stated in the results. 3.4. Quantitative bacteriology and histopathology Mice were euthanized by asphyxiation with CO2 and necropsied at day 5 post exposure. For enumerating viable bacteria in the lungs, spleens and livers of infected mice, the organs were removed aseptically, cut into small pieces, then homogenized using an aerosol-proof homogenizer, and plated as described previously [11]. For histopathological

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