INFECTION AND IMMUNITY, Sept. 1991, p. 2922-29280019-9567/91/092922-07$02.00/0

Live Vaccine Strain of Francisella tularensis: Infection andImmunity in Mice

ANNE H. FORTIER,'* MICHAEL V. SLAYTER,2 ROBERT ZIEMBA,'MONTE S. MELTZER,' AND CAROL A. NACY'

Departments of Cellular Immunology' and Comparative Pathology, 2 Walter Reed ArmyInstitute of Research, Washington, D.C. 20307-5100

Received 16 November 1990/Accepted 27 May 1991

The live vaccine strain (LVS) ofFrancisella tularensis caused lethal disease in several mouse strains. Lethalitydepended upon the dose and route of inoculation. The lethal dose for 50% of the mice (LD50) in four of sixmouse strains (A/J, BALB/cHSD, C3H/HeNHSD, and SWR/J) given an intraperitoneal (i.p.) inoculation was

less than 10 CFU. For the other two strains tested, C3H/HeJ and C57BL/6J, the i.p. log LD5* was 1.5 and 2.7,respectively. Similar susceptibility was observed in mice inoculated by intravenous (i.v.) and intranasal (i.n.)routes: in all cases the LD50 was less than 1,000 CFU. Regardless of the inoculation route (i.p., i.v., or i.n.),bacteria were isolated from spleen, liver, and lungs within 3 days of introduction of bacteria; numbers ofbacteria increased in these infected organs over 5 days. In contrast to the other routes of inoculation, miceinjected with LVS intradermally (i.d.) survived infection: the LD50 of LVS by this route was much greater than105 CFU. This difference in susceptibility was not due solely to local effects at the dermal site of inoculation,since bacteria were isolated from the spleen, liver, and lungs within 3 days by this route as well. Thei.d.-infected mice were immune to an otherwise lethal i.p. challenge with as many as 104 CFU, and immunitycould be transferred with either serum, whole spleen cells, or nonadherent spleen cells (but not Ig+ cells). Avariety of infectious agents induce different disease syndromes depending on the route of entry. Francisella LVSinfection in mice provides a model system for analysis of locally induced protective effector mechanisms.

Francisella tularensis, a facultative intracellular bacte-rium, initiates a potentially fatal human disease called tula-remia. Disease manifestation depends upon the route ofinoculation and ranges from ulceroglandular infection (entrythrough the skin) to respiratory tularemia (inhalation) to atyphoidal disease (unknown route of entry). Regardless ofthe initial site of infection, pneumonia is a common sequelaof tularemia (13). Survival ensures lifelong immunity toreinfection (20). A relatively avirulent Francisella strain, thelive vaccine strain (LVS), is used as a vaccine in humans,albeit with only variable effectiveness, to protect againstvirulent F. tularensis (4, 22). This vaccine strain itself,however, is fully pathogenic for certain animals and causes alethal infection in mice that is indistinguishable from humandisease (1, 6). Thus, the mouse is an extremely useful animalmodel for analysis of host response during tularemia.

In early studies with LVS, cells and serum from immuneanimals were transferred to nonimmune recipients to assessthe role of each in protection against virulent F. tularensischallenge. These studies suggest that LVS-immune cells, notserum, mediate protective immunity against infections withwild-type Francisella strains (7, 21). Two questions, how-ever, remain unresolved: the cells involved in passive trans-fer of immunity to wild-type F. tularensis and the relativeroles of antibody and cellular responses in primary infectionwith F. tularensis LVS. In this study, F. tularensis LVSinfection in mice was characterized not as a vaccine, but asa lethal infection caused by an intracellular pathogen. Sev-eral mouse strains were surveyed for susceptibility to dis-ease initiated by different routes of inoculation. The objec-tive of these studies was to characterize an experimentalanimal model of infection for analysis of cellular interactions

* Corresponding author.

that might lend themselves to immune intervention in tula-remia.

MATERIALS AND METHODS

Animals. Specific-pathogen-free BALB/cHSD and C3H/HeNHSD male mice were purchased from Harlan SpragueDawley, Frederick, Md., and used at 5 to 7 weeks of age.Pathogen-free C57BL/6J, A/J, C3H/HeJ, and SWR/J malemice were purchased from Jackson Laboratories, Bar Har-bor, Maine, and also used at 5 to 7 weeks of age. Mice werehoused in a barrier environment at Walter Reed ArmyInstitute of Research.

In conducting the research described in this report, weadhered to the Guide for Laboratory Animal Facilities andCare as promulgated by the Committee of the Guide forLaboratory Animals and Care of the Institute of LaboratoryAnimal Resources, National Academy of Sciences, NationalResearch Council.

Bacteria and growth conditions. F. tularensis LVS (ATCC29684; American Type Culture Collection, Rockville, Md.)was cultured on cysteine heart agar plates with 5% defibri-nated rabbit blood (Remel Labs, Lenexa, Kans.) for 4 daysat 37°C in 5% CO2 and at 95% humidity. Colonies wereselected for growth in modified Mueller-Hinton broth (Difco,Detroit, Mich.) supplemented with ferric PPi and IsoVitaleX(Becton Dickinson, Cockeysville, Md.) (2). Inoculated brothcultures were incubated for 2 days until the bacterial densityreached 108 to 109 CFU/ml. Aliquoted bacteria were frozenat -70°C. Aliquots (1 ml) were periodically thawed, andviable bacteria were quantified by plating serial dilutions in0.1% gelatin-saline on cysteine heart agar plates. CFU afterthawing were well within a 5% variation over a 4-monthperiod. Escherichia coli BORT (gift of Alan Cross, WalterReed Army Institute of Research) was inoculated into mice

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at a lethal dose according to previously published protocols(5).Animal inoculations. Mice were given different doses of

LVS at various anatomical sites. The lethal dose for 50% ofthe mice (LD50) was calculated for each route by the methodof Reed and Muench as discussed by Lennette (11). Toevaluate resistance to reinfection, we inoculated mice thatsurvived primary Francisella infection with an intraperito-neal (i.p.) dose of LVS that killed 100%o of naive mice.Enumeration of bacteria in tissues. Organs were aseptically

removed from infected mice and placed in gelatin-saline onice. Organs were homogenized in a Ten Broek tissue grinder,serially diluted, and plated on cysteine heart agar plates.Colonies were counted after 4 days of incubation at 37°C in5% CO2.

Adoptive transfer of resistance. Spleens from Francisella-immune or uninoculated mice were aseptically removed, andthe tissue was disrupted through a sterile 50-mesh stainlesssteel screen into Dulbecco modified Eagle medium (GIBCO,Grand Island, N.Y.) with 10% heat-inactivated fetal bovineserum (Hyclone). Erythrocytes were lysed with ammoniumchloride. Spleen cells were adjusted to 2 x 107 cells per ml.For certain experiments, single-spleen-cell suspensions wereincubated in tissue culture flasks for 2 h at 37°C in 5% CO2.Nonadherent cells were removed, washed in fresh medium,counted, and adjusted to 2 x 107 cells per ml. Immune cellswere treated with anti-CD4 (2RL) and anti-CD8 (83.12.5)antibody (a generous gift from Urszula Krzych, Walter ReedArmy Institute of Research) at 4°C and then lysed withguinea pig complement (Low-Tox M, Cederlane, Iowa) at37°C. Dead cells were removed by Ficoll gradient separa-tion, viable cells were washed, and cells were adjusted to 2x 107/ml. Cells were stained for surface immunoglobulin M(IgM) with a fluorescein isothiocyanate conjugate of goatanti-mouse IgM (Southern Biotech Corp., Birmingham,Ala.) and analyzed for fluorescence in a FACSCAN (BectonDickinson, South San Francisco, Calif.). A 1-ml volume ofwhole-spleen-cell suspension or nonadherent-cell suspen-sions (treated and untreated), was inoculated i.p. into syn-geneic mice, and mice were challenged with LVS within 2 hof cell transfer.To obtain serum for passive transfer, we sacrificed groups

of mice by exsanguination. Pooled blood from 5 to 10 miceper group was clotted, and serum was collected. Francisellareactivity of immune serum was analyzed by LVS enzyme-linked immunosorbent assay (ELISA) with glutaraldehyde-fixed bacteria. The subclass composition of serum Ig wasdetermined by coupling the LVS ELISA with goat anti-mouse subclass antibodies (IgM, IgGl, IgG2a, IgG2b, IgG3,and IgA) directly conjugated to horseradish peroxidase(Southern Biotech). Serum titers were also determined byslide agglutination of Francisella cells. Specificity was con-firmed by the absence of Escherichia coli agglutinatingantibody. A 1-ml volume of immune serum was inoculatedi.p. into syngeneic mice; control mice received 1.0 ml ofserum from normal, uninfected mice. Mice were challengedwith LVS within 2 h of serum transfer.

RESULTS

Francisella infection in mice: effect of route of inoculation.Six mouse strains were selected for determination of LD50 ofF. tularensis LVS by i.p. inoculation (Table 1). If theinfection was lethal, death was rapid: animals were sick byday 3 and moribund by day 5, and all animals were dead byday 7, regardless of the mouse strain. The animals that

TABLE 1. Susceptibility of inbred mice to i.p. infectionwith F. tularensis LVSa

Mouse ~Bacterial Mortality Logstrain inoculum ratio'LD"(CFU)

A/J 100 1/510' 3/5102 5/5 0.3103 4/4104 5/5

BALB/c 10-1 2/3100 6/8101 7/8 0102 8/8103 8/8

C57BL/6J 100 0/5101 0/5102 2/5 2.7103 4/5104 5/5

C3H/HeN 10-1 1/5100 3/5101 4/5 0102 5/5103 5/5

C3H/HeJ 100 0/310' 0/3 l102 3/3103 3/3

SWR/J 100 2/510' 3/5 0.4102 J40.103 5/5

a Mice were inoculated i.p. with 10-fold dilutions of LVS in a total volumeof 1.0 ml of gelatin-saline.

I Mortality ratio, number of animals dead/total number of animals inocu-lated.

survived infection for 10 days remained alive through 30days. The LD50 for four of the six mouse strains tested wasapproximately 1 CFU (range, 1 to 5 CFU, n = 5). Modestgenetically based resistance was suggested by the LD50 forCS7BL/6J mice, which was 700 organisms, and that forC3H/HeJ mice, which was 50 organisms.To determine the primary site(s) of bacterial replication

in susceptible strains, we removed various organs fromBALB/c or C3H/HeN mice inoculated i.p. with 103 LVSorganisms. Bacteriological examination of the tissues re-vealed early involvement of reticuloendothelial organs:spleen, liver, and lungs (Fig. 1). There was a logarithmicincrease in the number of viable LVS cells cultured fromthese tissues over time. LVS infection of these organspersisted until the death of the animal. Examination of thetissues revealed pathological changes in both the spleen andthe liver: necrotic foci with inflammatory infiltrates, primar-ily neutrophils and monocytes, were evident. Organismswere not found in other tissues examined (heart, stomach,kidneys).The two most susceptible mouse strains, C3H/HeN and

BALB/c, were infected by intravenous (i.v.) inoculation ofLVS (Table 2). Similarly to i.p. infections, animals givenhigh doses of LVS i.v. were sick by 3 days, moribund by 5

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A1o8

106

n0m0

0,

102

10 1

100

io8

106

c

0

IL

m

0,

102

10 1

1000 1 2 3

Days After Infection

C108

,06

cna0

0

0

10 4

1o 2

10 1

1 00

Days After Infection

D108

106

cn0

0

IL

0,

0

15

go

103

102

101

1002 3 4 5 6

Days After Infection Days After Infection

FIG. 1. Groups of mice were inoculated with 103 LVS by the i.p. (A), i.v. (B), i.n. (C), and i.d. (D) route. Three mice were sacrificed dailyafter inoculation, and the spleen (0), liver (0), and lungs (U) were removed and homogenized in gel-saline. Tissue homogenates were seriallydiluted in gel-saline and plated on duplicate CHA plates, and the number ofCFU per organ was calculated. The mean organ count from threemice is plotted.

days, and dead by 7 days. Again, animals that survived for10 days also survived through 30 days. The LD5sO for thesetwo mouse strains were not as low as those for i.p. inocula-tion: both C3H/HeN and BALB/c mice survived challengewith 100 CFU, whereas both strains were susceptible to <10

organisms given i.p. (Tables 1 and 2). Bacteriological exam-ination of the tissues from infected animals again indicatedthat the target organs were the liver, spleen, and lungs (Fig.1). The liver and spleen were colonized early. Lungs con-tained bacteria by 3 days. Over the next 7 days, bacterial

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TABLE 2. Susceptibility of BALB/c and C3H/HeN to infectionwith LVS administered by different routesa

Mouse Route of Bacterial Mortality Logstain inoculation inoculum ratiobLD5"(CFU)

BALB/c i.p. 100 3/5

I.v.

i.n.

i.d.

C3H/HeN I.p.

i.v.

i.n.

i.d.

lo'102103

100lo'102103104105106

lo'102103104

104105106107

101lo'102103

100lo'102103104

101lo'102103104105

10410'

106107

4/55/55/5

0/5 E1/41/42/42/33/44/4

0/50/55/5

0/50/51/5

3/5 14/5 1

1/40/53/41/44/44/4

0/50/53/55/55/5

0/50/55/55/5

0

2.5

2.5

>7.0

a Mice were inoculated with 1.0 ml of gel-saline suspension of bacteria i.p.,0.2 ml i.v., 0.05 ml i.n., and 0.1 ml i.d.

b Mortality ratio, number of mice dead/total number of mice inoculated.

counts in the liver and lungs increased. Bacterial counts inthe spleens of infected animals 7 days after inoculation (atime at which animals were moribund) actually decreased.Histopathological examination of the infected tissues againshowed inflammatory lesions that progressed from acute tonecrotizing over time. These lesions, however, were dif-ferent from those resulting from i.p. inoculation: the lesionswere granulomatous by 7 days.To simulate the natural route of infection by aerosolization

in the two most susceptible strains, we anesthetized C3H/HeN and BALB/c mice and administered LVS intranasally(i.n.) in a total volume of 50 ,ul. As with the other two routesof inoculation, animals were sick by 3 days, moribund by 5

TABLE 3. Infection of reticuloendothelial organs: time coursefor isolation of bacteria

Time for isolation of

CFU/organ Organ bacteria after inoculationa:i.p. i.v. i.n. i.d.

103 Spleen 1.2 1.2 3.6 2.0Liver 0.9 0.6 3.2 2.5Lung 2.1 2.3 1.6 >5

i10 Spleen 1.6 1.7 4.3 2.5Liver 1.2 0.9 3.7 2.8Lung 2.4 3.2 1.8 >5

105 Spleen 2.0 2.5 4.7 3.5Liver 1.7 1.7 4.3 3.0Lung 2.6 5.0 2.0 >5

a Time taken (days) after inoculation of mice to obtain 103, 104, or 10'bacteria from organs of C3H/HeN mice. Data were derived from bacterialcounts in organs as shown in Fig. 1.

days, and dead by 7 days. The LD50 of LVS given by thisroute in C3H/HeN mice was approximately 1,000 CFU,which is higher than that by i.p. or i.v. challenge. The LD50for BALB/c mice by this route was 100 CFU (Table 2).

Microbiological examination of the tissues from inocu-lated mice revealed bacteria in the spleen, liver, and lungs by3 days after inoculation (Fig. 1). These organs remainedinfected for 8 days after inoculation. Bacteria were notisolated from kidney, heart, or stomach tissues. Grosspathological examination of i.n. inoculated animals indicatedpulmonary involvement: all affected mice developed dys-pnea by 3 days. Histopathological examination of lung tissueshowed that bronchopneumonia was evident by 3 days andbecame necrotizing at 6 days. At 6 days, livers had foci ofacute inflammation with necrosis that became granuloma-tous by 8 days. At 6 and 8 days, spleens were enlarged andthere was significant proliferation of mononuclear cells inperiarteriolar lymphoid sheaths.The other natural route of human infection is through the

dermis. To determine whether mice were susceptible to LVSgiven by this route, we inoculated three mouse strains,C57BL/6J, C3H/HeN, and BALB/c, with a dose of LVS thatwas fatal by any other route of inoculation. All animalssurvived an intradermal (i.d.) inoculation (base of the tail)with 10' LVS cells. We attempted to calculate LD50s for twoof the strains, C3H/HeN and BALB/c, but fewer than 50% ofthe BALB/c mice died after an i.d. inoculation of 107organisms. C3H/HeN mice were more susceptible to thisroute, since the LD50 was 105'5 (Table 2).

It was possible that animals survived i.d. inoculationbecause the bacterium did not metastasize from the site ofinoculation. Organ distribution of LVS in i.d.-inoculatedmice, however, did not differ from that found after otherroutes of inoculation: bacteria were isolated from the spleen,liver, and lungs (Fig. 1). Additionally, initial growth of thebacterium within these tissues was not different from thatseen by the other routes. The only differences of note werethe times for these organs to acquire a particular number ofbacteria (Table 3). This time difference was most apparent inthe lungs of i.d.-inoculated mice. By 3 weeks after infection,all tissues of i.d.-inoculated mice were free of bacteria.

Resistance to reinfection. Mouse strains that will die afteri.p. inoculation of approximately 1 CFU of LVS (BALB/cand C3H/HeN) can withstand a dose of >100,000 CFU given

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TABLE 4. Protection against F. tularensis LVS infection bypassive transfer of serum or cells

No. of survivors/totalMice treated witha: no. of mice

challenged withb:

LVS E. coliSerum Cells (104 LD50) (10' LD50)Expt INone None 0/5 0/5Normal None 0/5 0/5Immune None 5/5 0/5None Normal spleen 0/5 0/5None Immune spleen 5/5 0/5None Immune spleen nonadherent 5/5 0/5

Expt IINone None 0/5None Normal spleen 0/5None Immune spleen 5/5None Immune spleen nonadherent 5/5None Immune spleen Ig+ 0/3

a Donors were either C3H/HeN mice inoculated with 103 LVS cells at thebase of the tail 2 weeks before collection of serum or spleen cells or age- andsex-matched normal controls. Recipient mice were age- and sex-matchedC3H/HeN mice. Serum and cells were obtained and characterized as de-scribed in Materials and Methods.

b Recipients were challenged i.p. 2 h after passive transfer of cells or serumand were observed for up to 30 days.

at an i.d. site. To determine whether resistance to infectionobserved after i.d. inoculation is mediated by immunologicalmechanisms, we measured the protection induced by i.d.inoculation for resistance to a second infectious challengegiven i.p. All five mice (100%) of each strain inoculated i.d.with 103 LVS cells 2 weeks earlier survived an i.p. challengewith LVS at a concentration (104 LVS cells) that killed allcontrol mice. Control mice died within 7 days; i.d. immu-nized mice were still alive after 30 days. C57BL/6J micewere also tested for resistance to rechallenge with LVS.Mice of this strain were also 100% protected from anotherwise lethal i.p. disease by prior i.d. LVS inoculation.

Transfer of humoral immunity. Survival of i.p. inoculationby i.d. infected mice suggested that specific immune mech-anisms were induced during i.d.-initiated disease. To assessthe production of antibodies to LVS, we collected serumfrom i.d. inoculated mice for at least 14 days and up to 30days after infection. These serum samples agglutinated apreparation of LVS, but not unrelated gram-negative bacte-ria such as E. coli or Shigella species. Serum titers ofagglutinating antibodies of individual i.d.-inoculated miceranged from 1:8 to 1:16. Serum samples from age- andsex-matched normal mice did not agglutinate LVS or E. coli.To determine whether protective immunity could be trans-

ferred with serum, we pooled serum samples from fiveC3H/HeN mice that had been inoculated i.d. with LVS (theanti-LVS pooled serum titer was 1:8 by agglutination) andtransferred the serum i.p. to normal, uninoculated syngeneicmice. Relative concentrations of Ig classes in the pooledimmune serum were determined by LVS ELISA. The anti-LVS ELISA titer of whole serum was 1:10,240, and specificLVS-binding titers by subclass were as follows: IgM, 1:640;IgGl, 1:80; IgG2a, 1:2560; IgG2b, 1:1,280; IgG3, 1:40; IgA,not detectable. This serum, but not serum from uninoculatedanimals, protected mice from lethal i.p. challenge with LVS.The protective effects were specific, and mice treated withthis serum did not resist challenge with an antigenicallyunrelated microorganism (Table 4).

Transfer of cellular immunity. To assess the role of cells inprotection against primary infection with LVS, we trans-ferred spleen cells from i.d. inoculated mice into naiveanimals. Recipient mice were challenged with a lethal doseof LVS within 2 h of cell transfer. The concentration of allspleen cell preparations was 2 x 107 cells per mouse. Wholespleen cells and nonadherent spleen cells from i.d. inocu-lated mice, but not normal (uninoculated) mice, transferredprotection against lethal i.p. challenge with LVS, but notagainst challenge with an unrelated gram-negative organism(Table 4). To further characterize the protective cells in thistransfer system, we treated immune spleen cells with acocktail of anti-Thyl.2, anti-CD4, and anti-CD8 antibodiesand complement to remove T cells. The resulting cells,found to be 82% surface Ig+ by FACS analysis, were unableto protect mice from lethal LVS infection (Table 4).

DISCUSSION

There are a number of infections with the same microor-ganism that appear as distinct diseases depending on the siteof pathogen inoculation. Cutaneous disease is generallyinitiated by direct contact at a skin site. Systemic infectionsresult either from spread of the agent via vascularization andhematological dissemination to other organs, or from directinfection (in the lungs, for instance, by inhalation of theinfectious agent). Sporotrichosis, a fungal disease caused bySporothrix schenckii, has three distinct forms depending onthe route of inoculation: a cutaneous lesion develops at thesite of skin contact with spores, a chronic pneumoniadevelops as a result of inhalation of spores, and a dissemi-nated form can result from lymphatic or hematologic spreadfrom either of these primary sites (19). Bacillus anthracis thebacterium responsible for anthrax, causes a cutaneous infec-tion if inoculation is through the skin, but a pulmonaryinfection if the route of inoculation is by inhalation of spores(8). Brucellosis, erysipeloid, glanders, plague, and tularemiaall have cutaneous manifestations as well as systemic formsof disease (14, 18, 19, 23). In human tularemia, the usualroute of entry is through the skin, which leads to develop-ment of ulceroglandular disease. There is lymph node in-volvement, and dissemination can occur via the blood. F.tularensis may also be transmitted via aerosols to the lungs.This route of inoculation leads to pulmonary disease that, ifuntreated, is fatal in up to 60% of cases. The ulceroglandularform of the disease has a fatality rate of only 5% if untreated(19, 20). The mechanisms responsible for these differencesbased on route of inoculation have not been elucidated forany of the infectious diseases described above. It is still notknown which local mechanisms, specific or nonspecific, playa role in protection. If the host is competent to mount aneffective immune response, why are these mechanisms noteffective during systemic disease?

F. tularensis LVS infection in mice is similarly dependenton the route of inoculation. i.p., i.v., and i.n. inoculation alllead to early reticuloendothelial involvement (1 to 3 days)and bacterial growth within tissues of the lungs, liver, andspleen. There appear to be some minor differences in thehost response to challenge by these different routes. Bacte-rial growth slows, and there is late (after 7 days) granulomadevelopment following i.v. and i.n. inoculation, but not i.p.inoculation. Eventually, however, the majority of the micedie after an initial inoculum of less than 1,000 CFU (Table 2).A dose that is lethal by any other route, however, is notlethal by the i.d. route. Perhaps a nonspecific protectivemechanism against LVS operates at the site of i.d. inocula-

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tion. Do the animals survive because the bacteria do notinfect or disseminate to tissues by this route? Our studiessuggest that this is unlikely: the same tissues (spleen, liver,and lung) become infected and support bacterial growth afteri.d. inoculation as those that are affected when the organismis inoculated by a route that leads to death. Althoughbacteria initially replicate in these organs just as they doafter a lethal challenge route, they are completely cleared by2 to 3 weeks after i.d. challenge. The other striking differ-ence between routes of inoculation is noted for the lungs:bacteria are detected in the lungs of i.d. inoculated mice onlyafter 5 days. Furthermore, the presence of the bacteria in thelungs is transient and may not indicate infection of thistissue, since numbers of recovered bacteria do not increasein this organ. Therefore, an effective and appropriate re-sponse that limits bacterial survival in the lungs was gener-ated by the host, and these i.d. inoculated animals survivedinfection. What mechanisms are at work i.d. that are notengaged when the organism enters the host by any otherroute?

Since the organism establishes an infection in the liver andspleen by 2 to 3 days in i.d. inoculations, just as in lethaldisease, the protective response must be initiated early. Theearliest event in i.d. inoculation is initial contact of thebacterium-with local skin cells. Of particular interest to thisstudy is the potential role of T cells expressing yB receptors(3). The potential importance of this particular T-cell lineagein the Francisella infection model is suggested by the stra-tegic location of -yb cells in the skin (10) and by the functionalabilities of these cells to recognize major histocompatibilitycomplex-linked antigens (12), produce lymphokines (17),develop cytotoxic potential (15), and react to bacterial heatshock proteins (17). T cells expressing yS receptors haverecently been implicated in development of the primaryimmune response to Mycobacterium tuberculosis, anotherintracellular organism (9). The characterization of this route-dependent variation in development of immunity to Fran-cisella infection in C3H/HeN mice will enable us to examinethe role of -yb T cells in tularemia.

Passive transfer of immunity to lethal LVS infections withboth serum and cells (Table 4) suggests that protective hostresponses to this vaccine strain during primary disease differfrom those that influence resistance to the wild-type Fran-cisella strain. In earlier studies that focused on LVS as avaccine rather than a pathogen, only cells from vaccinatedanimals were capable of adoptively transferring immunity tovirulent F. tularensis (7, 21). Here we demonstrate thatserum is quite effective in protecting mice from i.p. LVSinfections, and we recently generated a monoclonal antibodyreactive with LVS that also protects mice from lethal disease(16). That cells can passively transfer protection againstlethal i.p. LVS infections was suggested by earlier studiesinvolving challenge with the virulent wild-type pathogen (7,21). The nature of the protective cells effective in theseearlier studies was never determined. We find that thetransfer of immunity to LVS infection by spleen cells is farmore complicated than anticipated: whole immune spleencells and nonadherent immune spleen cells, but not B cells,passively transferred protection, and this protection wasspecific for Francisella species (Table 4). However, a num-ber of our experiments (data not shown) suggest that pro-tection is not mediated by the classic CD4 T-cell componentof immune spleen cells. We have yet to determine theprecise phenotype of the protective cells in our system, butthese studies are under way.By simply changing the route of entry, it is possible to

analyze both protective immune mechanisms and ineffectiveresponses induced in a single animal strain by a singlebacterial strain. F. tularensis LVS infection in mice providesa model system with which to analyze these host responses.Mice susceptible to the lethal effects of F. tularensis LVSinfections clearly possess the machinery to generate anappropriate and protective immune response. For unknownreasons, the machinery is fueled only after i.d. exppsure tothe pathogen.

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