INFECT-ION AND IMMtjNITY, May 1972, p. 734-744Copyright li; 1972 American Socicty for Micr-obiology

Vol. 5, No. 5Prinzted in U.S.A'.

Pathogenesis of Tularemia in Monkeys AerogenicallyExposed to Francisella tularensis 425

ROBERT L. SCHRICKER,l HENRY T. EIGELSBACH, JOHN Q. MITTEN,2 AND WILLIAM C. HALL'U.S. Army Biological Defense Research Laboratory, Fort Detrick, Frederick, Marylanfd 21701

Received for publication 14 February 1972

The pathogenesis of tularemia was studied in groups of rhesus monkeys (Macacamulatta) that inhaled graded 10-fold doses ranging from 10 through 106 organismsof Francisella tularensis 425, a strain highly virulent for the white mouse but ofreduced virulence for the domestic rabbit. Mean incubation periods ranged from 3to 6 days followed by acute illness lasting 5 to 11 days with subsequent recovery ofmost animals. The higher inhaled doses resulted in shorter incubation periods, longerand more severe acute illnesses, and 18% mortality at the highest dose. Strain 425multiplied in the lungs, disseminated to the regional lymph nodes, and became sys-

temic. Maximal bacterial populations in tissues were reached by the 7th day afterexposure of the animals regardless of the number of organisms inhaled. F. tularensiswas no longer recoverable from any of six tissues examined 2 months after exposure.

The most significant tissue changes occurred in the lungs; these consisted of foci ofliquefaction necrosis, lobular consolidation, and pleural effusion and adhesions. Thedata indicate that the inhaled dose of strain 425 determined the maximal growth ofthe organism in the lungs which in turn influenced the severity of the usually self-limiting pneumonia and systemic infection. Although the monkey is less resistantto tularemia than is man, this laboratory animal when infected with F. tularensis 425provides a useful model for the self-limiting type ofhuman pulmonary tularemia usu-

ally observed in Europe and Asia but to a lesser extent in North America.

It is not generally recognized that two majortypes of tularemia occur in North America al-though this fact was reported years ago (1, 11).Each of the two types is characterized by an epi-demiologic pattern and virulence of the infectingFrancisella tularensis strain. Strains of high viru-lence for man, designated type A by Jellison et al.(11), are usually associated with tick-borne tula-remia of rabbi:s (Langomorpha). Nearly 90% ofhuman cases reported in North America are of thistype. Strains of lowered virulence for man (typeB) are usually associated with the water-bornedisease of rodents (Rodentia). They are respon-sible for only 5 to 10%, of reported human casesin North America (8) although many cases areprobably undiagnosed (1). However, a major out-break of relatively mild tularemia was recentlyreported among muskrat trappers in Vermont (3,10, 26). The morbidity of the disease was com-parable to that most frequently observed in Eu-

1 Prescent address: Veterinarly Biologics-V/S APHS, Hyatts-ville, Md. 20782.

2 Present address: Dept. of Pathology, School of Medicine,Johns Hopkins Univ., Baltimore, Md. 21205.

3 Present address: 140 Pinecrest, Saln Antonio, Tex. 78209.

rope and Asia where it is sometimes called pale-arctica tularemia (15, 16).The pathogenesis of fatal tularemia resulting

from type A fully virulent F. tularensis strainSCHU S4 (4) and the immuLnogenicity againstit produced by live vaccine prophylaxis withstrain LVS (5) have been well documented forlaboratory animals including the monkey (7, 13,23, 24, 25); controlled vaccine evaluation andtherapy studies have also been reported for man(9, 19, 20, 21). The purpose of the present studywas to characterize the pathogenesis of type-Btularemia that is usually nonfatal for man. Therhesus monkey (Macaca mulatta) was chosen asthe test animal because of similarities in tularemicinfection in man and monkey (6). F. tularensis425 (2) was selected for this study because ourprevious experience, as well as that of others(18), had shown it to have the virulence and cul-tural characteristics of most Eurasian strainsas well as of strains involved in the Vermontoutbreak in contrast to those of strain SCHUS4. Several F. tularensis strains were studied withregard to fermentative characteristics and viru-lence for mice and rabbits. The infection was

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PATHOGENESIS OF TULAREMIA IN MONKEYS

evaluated by determining and correlating (i)the clinical course of the illness and (ii) bacterialdissemination, multiplication, and persistence aswell as (iii) the character and course of theanatomic pathologic process.

MATERIALS AND METHODS

F. tularensis strains. The origin of strains 425 (2),503 (16), and SCHU S4 (4) have been previously re-ported. We isolated strain Vt 68 from a sample ofmuskrat spleen from an animal trapped during the1968 Vermont outbreak (26); the sample was pro-vided by Lowell S. Young of the National Center forDisease Control, U.S. Department of Health, Educa-tion, and Welfare, Atlanta, Ga. All organisms werecultivated in a modified casein partial hydrolysateliquid medium (5). Cells harvested after 18 hr of incu-bation with shaking at 37 C contained approximately3 X 1010 viable organisms per ml determined by acolony count on glucose-cystein-blood-agar (GCBA)(5). Median subcutaneous lethal doses for white mice,20 mice per group, and domestic rabbits (Oryctolagus),6 animals per group, as well as glycerol fermentationwere used to distinguish type A from type B (1, 15,16). Type A strains of F. tularensis ferment glyceroland require less than 10 organisms to kill rabbits inocu-lated subcutaneously, whereas strains of type B do notferment glycerol and require more than 106 organismsadministered subcutaneously to kill rabbits.

Monkeys. Rhesus monkeys were caged individuallyand conditioned for 6 months before use. Animalsweighed between 2 and 3.5 kg and were randomlydistributed by weight and sex.

Aerogenic exposures. Strain 425 aerosols were gen-erated in a large sphere from liquid suspensions with anebulizer producing particles primarily in the range of1 to 5,um in diameter. The aerosols were allowed toequilibrate at 21 C and 85% relative humidity beforethe animals were exposed. Equipment and methodsfor generating static aerosols, determining inhaleddoses, and maintaining whole-body exposed animalshave been described by Jemski and Phillips (12).

Clinical study. One group of 10 monkeys inhaledapproximately 10 organisms, and other groups of 20to 30 monkeys inhaled 102, 103, l04, 105, or 106 cells.Monkeys were examined daily for signs of disease(exemplified by acute illness) and 2 to 3 times a weekthereafter until the studies were terminated 120 daysafter exposure. Body weights were determined at ap-proximately weekly intervals throughout each study,and data were calculated as per cent change from pre-exposure weight.

Standard clinical laboratory determinations madeon heparinized venous blood samples of all monkeysincluded plasma C-reactive protein (CRP), correctederythrocyte sedimentation rate (ESR), and erythrocytepacked-cell volume (PCV). Tests were made on mon-keys at 2- to 4-day intervals through the acute illnessand at 35, 60, 90, and 120 days after challenge. Baseline values were established over a 3-week period be-fore exposure. Blood-urea-nitrogen (BUN) and cre-atinine (Auto Analyzer, Technicon Co.), as well asserum glutamic-pyruvic transaminase (SGP-T, Sigma

Chemical Co.), values were determined on serumsamples of 10 of the monkeys inhaling 105 organisms.

Serum F. tularelnsis agglutinin titers (5) were deter-mined on all animals before exposure and on survivors14, 35, 60, 90, and 120 days after inhaling strain 425.

Chest roentgenograms were made of approximatelyone-half of the monkeys from each group (5 to 15animals) before and at 2- to 4-day intervals throughthe 2nd week after exposure and again at 3 and 4 weeksor until there was no longer radiographic evidence ofpulmonary disease. Roentgenographic classification ofpulmonary lesions and other clinical criteria for classi-fying the severity of the illness are summarized inTable 1.

Bacteriological and pathological studies. Groups of36 monkeys each were administered 104 or 106 cellsof strain 425 via the respiratory route in the mannerpreviously described. Two to four previously assignedmonkeys from each group were to be killed (Nembutal,Abbot Co.) and necropsies to be performed 1, 3, 6,10, 15, 21, 35, 60, and 120 days after challenge.Moribund animals were killed and examined; animalsfound dead were necropsied immediately. Samples oftissues were removed aseptically from the lungs,tracheobronchial lymph nodes, spleen, liver, femoralbone marrow, and blood. Tissues, except blood, wereweighed and then ground in ten Broeck tissue grinderscontaining 2 ml of sterile 0.1% gelatin-saline solution.Blood and dilutions of tissue suspensions were platedon GCBA and incubated at 37 C; colonies werecounted after 96 hr. Weights of the lungs, spleen, andliver were recorded. Viable strain 425 populationswere reported on the basis of an entire organ, pergram of bone marrow or lymph nodes, and per milli-liter of blood.

At the necropsy periods designated above, two tofour monkeys from each group were examined formacroscopic and histologic lesions. Lungs were in-flated with 10% buffered Formalin instilled throughthe trachea which was then ligated, and the specimenwas submerged in Formalin. Samples of tracheo-bronchial lymph nodes, liver, spleen, and any otherorgans and tissues that appeared abnormal macro-scopically were also preserved in Formalin. Fixedtissues were embedded in paraffin, cut into 5- to 7-,umsections, and stained by hematoxylin-eosin and Geimsamethods (13). Chest roentgenograms were made ofone or two of the monkeys from each group at eachtime period for comparison with necropsy observa-tions.

RESULTSStrain comparison. Virulence and glycerol

fermentation data of four strains of F. tularensisare shown in Table 2. Strains 425, Vermontstrain VT 68, and Eurasian strain 503 producedcomparable data. These three strains differedmarkedly in rabbit virulence and glycerol fer-mentation from North American strain SCHU S4.

Clinical. Clinical data on monkeys inhaling10-fold doses ranging irom 10 to 106 cells ofstrain 425 are shown in Table 3. All animalsbecame infected. Group mean incubation periods

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TABLE 1. Criteria for classifying severity of tularemia ini monzkeys

Severity of illness'Parameter

Mild Moderate Severe

Temperature, rectal. Febrile to 3 days, maxi- Febrile to a week, maxi- Febrile longer than a

mum > 103.5 F (ca. mum > 104.0 F (ca. week, maximum >39.7 C) 40.0 C) 105.0 F (ca. 40.6 C)

Anorexia None or present I to 3 Present to a week Present longer than a

days weekWeakness, depression,

or both............. None Present to a week Present longer than a

weekBody weight ......No change or gain No change or ioss > 10%" loss

X ray, chest .......... No lesions or+ for to +1 to +3, lesions for +3 to +5, lesions 2 to 56 days only week or longer weeks

C-reactive protein.. Negative to +1 for 3 >+I for about a week, >+3 for longer than a

days maximum +3 weekErythrocyte sedimenta-

tion rate (mm/hrl) 15 for 1 to 3 days >15 for week or longer >25 for longer than a

week

a Roentgenographic classification of pulmonary lesions: +1, minimal infiltrate, diffuse or localized;+2, definite infiltrate, diffuse or localized (if diffuse-miliary or 1-cm bronchopneumonic patches; iflocalized-an ill defined 1- to 2-cm bronchopneumonic patch); +3, diffuse 0.5- to 1-cm or well defined2- to 3-cm bronchopneumonic patches; +4, multiple 1-cm infiltrates or early lobar consolidation (ani-mals exhibiting these lesions classified severely ill regardless of other data); +5, coalescence of theabove multiple infiltrate with or without extensive lobar consolidation, obscuring both mediastinumand heart shadow.

b Normal: <1 mm/hr corrected for packed cell volume.

TABLE 2. Virtule,ice anld glycerol fermentation of Franicisellca tuilarenisis strainis

Mouse LD,ao Rabbit LDso' GlycerolStrain Source (no. of organisms) (no. of organisms) fermented

425 Tick (U.S.A.) 1 >106 0Vt 68 Muskrat (U.S.A.) I >106 0503 Man (U.S.S.R.) 1 >106 0SCHU S4 Man (U.S.A.) 1 <10 +

a Subcutaneous inoculation.

TABLE 3. Franicisella tuilarenlsis 425 inftction in monlkeys following respiratory exposure

Per cent animals showing illness'Mean no.ofActilnsorganisms Incubation (days) duration (lays)

None Mild Moderate Severe Fatal

10 6 (4-10)b 5 (2-9)h 30c 40 30 0 0102 5.5 (4-7) 6.5 (1-17) 0 50 45 5 0103 4.5 (2-8) 9 (4-22) 0 57 40 0 3104 4 (2-7) 10 (4-18) 0 3 77 13 7105 3 (3-4) 11 (7-15) 0 0 80 12 8106 3 (2-5) 9 (3-19) 0 0 55 27 1 8

a Values based on groups of 20 to 30 monkeys, except that 10 animals only were administered 10 cells.6 Mean to nearest one-half day and range.c Based on development of diagnostic F. tularentsis serum agglutinin titers.

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.-.-.

...

FIG. 1. Serial chest roentgenograms of a monkey after inhaling 106 cells of Francisella tularensis 425. (A)Day 2, lung fields similar to pre-exposuire; note distinct cardiac and diaphragmatic borders as well as the uniformdensity of lung fields. (B) Day 6, nodular infiltrates in all lobes and extensive areas of bronchopneumonia in theleft middle lobe and in both lower lobes. (C) Day 14, multiple nodular infiltrates still present in both lungs, butareas of bronchopneumonia are less extensive than on day 6. (D) Day 21, marked clearing of infiltrates with onlya few small nodular lesions still present in both lungs. Twenty-eight days after exposure there was complete clearing.

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SCHRICKER ET AL. INFECT. IMMUNITY

Days After Challenge

RectalTemperature

(F)

| ~~~~Anore.Animols

with signs

Deprest c~~~~nd/,F ~~~~weckn,

Lung lesions(X-roy)

C-Reactiveprotein (mm)

Erythrocytesedimentationrate tmm/hr)

Body Wt. Change (%Deoths (%)

,.1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15

10 _ _

03 - s; - - - - - - _-l<104e--_--J' ** - - *

101

37 100 87 93 27 13 23

siaon

/orness

3 27 57 2713 10

A

32- v

2 - o2

-

2 -

2

40 -

30 -

2010 -

0

0 -S _

3 3-5

16 17 18 19 20 21 28 35601o0* * o * v , + * IL _ _ _l

o +1 +3 *8 +1S

FIG. 2. Clinical data of illnless produced in 30 Macaca mulatta by respiratory exposure to 104 orgaiiisms ofFrancisella tularensis 425 (meazi values).

ranged from 3 to 6 days and were inversely re-lated to the number of inhaled cells. The majorityof monkeys inhaling 103 to 106 cells had incu-bation periods of 3 to 4 days; those given 10 to102 cells had 1- to 2-day longer and less well-defined incubation periods. The mean durationof the acute illness was 5 to 6.5 days at the lowertwo doses and 9 to 11 days at the higher doses.The mean duration of illness at the highest dosewas shortened by the death of some animals.Severity of the acute illness was directly related toinhaled dose, generally mild at the 10 to 102 dosesand moderate to severe at the higher doses. Therewas significant difference (P < 0.05) in severityof the illness neither between those inhaling 102and 103 cells nor between those given 104 and 105organisms. Clinical recovery usually ensued; mor-tality rate was directly related to inhaled dose butdid not exceed 18% even when 106 organismswere inhaled.

Serial chest roentgenograms of a representativemonkey which inhaled 106 cells of strain 425 areshown in Fig. 1. Approximately one-half of themonkeys exposed to 103 or fewer cells showed nodetectable lesions. The lungs of animals inhalingeven the highest dose were generally free frominfiltrates through the third day. Maximal severityoccurred from day 6 to day 14. Lesions rangedfrom minimal diffuse infiltrates particularly atthe lower doses and increase in severity at thehigher doses to coalescence of multiple infiltrates

with extensive lobar consolidation. Two animalsinhaling 106 cells exhibited bilateral pneumo-thorax between days 10 and 14 and one of theselater developed a pneumatocele in the left lung.One animal given 105 cells developed a lung ab-scess. Resolution of lesions was generally com-plete 2 to 4 weeks after a dose of 10 to 101organisms and 4 to 5 weeks after inhalation of 105or 106 cells. Lung lesions observed on roentgeno-grams often indicated a more severe diseasethan signs suggested upon physical examina-tion; therefore, pulmonary lesions were a majorfactor in classifying severity of the illness.Roentgenographic and other clinical findingsin animals inhaling 104 and 106 organisms aresummarized in Fig. 2 and 3, respectively.Plasma CRP was detected while signs of infec-

tion were observed. The majority of the animalsinhaling 104 or more organisms were CRP-posi-tive within a day of the onset of fever and re-mained positive through the 10th to 15th day.Monkeys given 103 or fewer cells were oftenCRP-positive only once or twice after exposure.Peak levels occurred in animals exposed to alldoses between the 6th and 10th day and weremost marked in those with the most severe illness.Abnormal ESR values (>1 mm/hr, corrected)generally occurred during the same period CRPwas detected and showed a similar direct rela-tionship to severity of disease. CRP and ESR weregood acute-phase indicators, correlating well

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PATHOGENESIS OF TULAREMIA IN MONKEYS

Days after Challenge .1 0 1 2 3 4 5 6 7 8, 9 10 11 12113 1415 16 17 18 19 20;21 28 35 60120F ~ ..`Rectal

Temperature

(F)

Animalswith signs

Mol

Anorxe

Depressiand/or

weoakne

Lung lesions

(X-ray)

C-Reactiveprotein (mm)

Erythrocytesedimen ationrate (mm/hr)

Body Wt. Change l%Deaths (%)

105-

104 -

1 03 - - - - -

102

101 -

41 73 69 95 73 69 64 41 2718 9

ion 9 9 36 45 18 9 9r

4 -

3-

2-

0-c

4

30 -

20-10-

0 -7 -9 -11 -6 +1 +5 +149 9

FIG. 3. Clinical data of illness produced in 22 Macaca mulatta by respiratory exposure to 106 organisms ofFrancisella tularensis 425 (mean values).

with development and severity of lung lesionsand other signs (see Figs. 2 and 3); similar find-ings have been reported for man (17, 20). PCV re-mained normal (mean 41 %) in all animalsthroughout the studies. Creatinine remained atpre-exposure levels (mean 1.10 mg/100 ml) amongmonkeys inhaling 105 cells but BUN increasedfrom a mean of 20 mg/100 ml to 30 mg/100 mlfrom day 4 through day 10, suggesting very mildrenal impairment, increased protein catabolism,or both. SGP-T values among these same animalsrose from a mean of 25 Sigma Frankel (SF)units/ml to a borderline pathologic value of40 SF units/ml during the same period, suggestinga very mild hepatotoxic effect. These mild, re-versible, serum chemical changes are commonlyseen during acute infections.Body weights of the majority of monkeys

exposed to the three lower doses remained nearpre-exposure levels through the third week afterexposure and showed approximately 15% in-creases by the 17th week. Bodyweights of monkeysgiven the three higher doses were below pre-exposure values from the first through the thirdto fifth week after exposure, with maximal meanweight losses of 6% to 11% occurring 2 to 3 weeksafter exposure. Animals given the highest strain425 doses generally showed the greatest weightloss. Seventeen weeks after exposure, monkeys inthe three higher dose groups showed weight gainssimilar to those given the lower doses.

TABLE 4. Serum agglutinini responise of monkeysexposed aerogenically to Francisella

tularensis 425

NoI.of Reciprocal mean titer of survivors on indicated dayNorga- after exposureanismsinhaled

14 60 90 120

10 80 960 640 320 160102 160 960 480 320 320103 160 1,505 1,565 640 320104 160 3,105 1,472 640 640105 670 3,010 1,855 1,153 416106 2,305 3,167 2,996 1,790 992

a Values based on groups of 10 to 20 monkeys.

All monkeys exposed to strain 425 developedF. tularensis agglutinins. Maximal mean titerswere recorded on the 35th day and graduallydeclined thereafter (Table 4). The mean agglutinintiters were higher with increasing inhaled doses oforganisms.

Bacteriologic data. Strain 425 was rarely re-covered from tissues of monkeys longer than 4weeks after the animals inhaled 103 or fewer or-ganisms. The rates of growth of strain 425 in thelungs of monkeys receiving aerogenically 104 to106 organisms are shown in Fig. 4. Quantitativebacteriologic data from other tissues of some ofthese animals are shown in Table 5. Twenty-four

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SCHRICKER ET AL. INFECT. IMMUNITY

10

10o

10

10

10

* 040 ° 3

10 0

3

2

2E

10

I I LI

0 3 6 7 10 15 1 216 2

Days After Challenge

FIG. 4. Rate of growth of Franzcisella tularenisis 425 inl the luntgs of monzkeys afier inihalationi of 104 or 106 or-

ganisms. Meani anid inidividtual popullationzs represenited by solid aiid openi symbols, respectively.

TABLE 5. Viable Franicisella tularentsis in tissules of monikeys after aerogeniic admiinlistration of strailn 425

Organ-Tissue isms -- _

inhaled

~

Tracheobron- 104chial nodes 106

Spleen 10I

100

Liver 104106

Bone marrow 104106

Blood 104106

Logio no. of organisms on indicated day after exposure"

1 3

0,0 6,53,3 7,6

0,0 3,00,0 4,2

0,0 -0,00,0 118,4

0,0 0,0

0,0 0,0

0,0 0,00,0 0,2

6,6

0,34,5,4,6

5,45,7,5,6

0,00,2,1,2

0,03,4,1,4

7 10

8,7b 5,62,6 6

4,5 0,36,4 3

5,67,4

3,3

3,0

102,0

0,0

4

0,02

0,03

15

0

2,4

35,0

0

0,0

0

0,0

0

0,0

21 35

3 2,02,1 2

0

0,0

0

0,0

0

0,0

0

0,0

0,0

0

00

0

0,00

0,00

60 120

0,0

0

0,0

0

0,0

0

0,0

0

0,0

0

0,00

0,0

0

0,0

0

0,0

0

0,0

0

,t One to four monkeys examined on each indicated day. Data are based on results per organ for spleenand liver, per gram of lymphatic or bone marrow tissue, and per milliliter of blood. No recovery indi-cated by 0.

h Underlined values indicate animals moribund or dead.

hours after exposure, the viable strain 425 popula-tion in the lungs was generally as high as the in-haled dose and in some instances 10-fold higher.At this time, F. tularensis 425 was also recoveredfrom the tracheobronchial lymph nodes ofmonkeys inhaling 106 organisms but not fromcomparable tissues of monkeys given 104 organ-isms, indicating that the infection was still local-ized at the site of exposure in animals given thelow dose but regional in those given the highdose. On the third day, the strain 425 mean popu-lations in the lungs were approximately 50-foldhigher than inhaled doses, and the organism

was recovered from tracheobronchial lymph nodesof animals inhaling either the 104 or 106 dose. Atthat time, the spleen of 1 of 2 monkeys given 104organisms and the spleens of both monkeysgiven 106 organisms were positive for F. tularensis,indicating that systemic infection had occurred.Strain 425 was also recovered from the liver andblood of monkeys given 106 organisms on the3rd day. Thus, on the 3rd day the organism wasstill regional or of limited dissemination in mon-

keys given 104 cells, but it had become systemicin animals exposed to 106 organisms.The maximal viable bacterial population was

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PATHOGENESIS OF TULAREMIA IN MONKEYS

10 Cells

4.0

3.0

2.0

1.0

* 10 Cells

L1 3 6 7 10 15 2 5 60 120

Days after Exposure

FIG. 5. Ratios of monkey lung weight to body weight after inhalation of 104 or 106 organisms of Francisellatularensis 425.

reached in nearly all organs and tissues by thesixth to seventh day, regardless of number ofinhaled organisms. Mean maximal viable strain425 populations in the lungs were approximately1,000-fold higher than the inhaled dose. Lungsof animals dying during the acute infection gen-erally contained 108 to 1010 cells of strain 425,and all other organs assayed also contained largenumbers of the organism. The order and time ofclearance of organisms from tissues after expo-sure were: femoral bone marrow, liver, and blood,10 to 15 days; spleen, 15 to 21 days; lungs, 21to 35 days; and tracheobronchial lymph nodes,35 to 60 days. Bacteriologic data indicated thatdissemination and proliferation of organismswere more rapid and extensive and clearanceoccurred later in animals given the higher doses.

Pathologic observations. Gross lesions oftularemia were limited to the lungs in monkeysinhaling 101 or fewer cells. Lungs of these animalscontained a few scattered areas of congestion,lobular consolidation, and miliary necrotic foci.Lesions were rarely detected after the fourth tofifth week.

Tissue changes among monkeys inhaling104 cells and serially necropsied after exposurewere similar but more extensive than those inanimals exposed to lower doses of strain 425.Histologic lung lesions consisting of small areasof bronchopneumonia with mixed infiltrates ofpolymorphonuclear leukocytes, lymphocytes, andhistiocytes were detected from the 3rd to 6thday (early-type lesion). From the 6th through21st day, lesions progressed to focal liquefactivenecrosis surrounded by histiocytic infiltrates(middle-type lesion). Partial to complete clear-ing of the abscess, either by emptying of puru-lent material into a bronchus or by organi-

zation with early fibrosis (late-type lesion), wasobserved from days 15 through 35. Mixturesof these types of lesions were usually found asnew foci of infection developed and early lesionsresolved. The earliest pulmonary lesions detectedgrossly were diffuse reddening and foci of atelec-tasis; these were observed on day 6. Lesions onday 10 consisted of multiple lobular consolida-tion that became more diffuse through day 15and sometimes remained until day 35. Only afew pleural adhesions were detected later. In thetracheobronchial lymph nodes, mixed cell in-filtrates and liquefactive necrosis were detectedfrom days 6 to 15, followed by lymphoid andreticular cell hyperplasia from days 15 to 21.Histologic liver changes occurred between the6th and 35th days and consisted of fatty changes,circumscribed areas of mononuclear infiltrates,mixed inflammatory cells in the sinusoids, andenlarged Kupffer cells. Mild centrilobular de-generation was seen at all necropsy periods butwas probably unrelated to tularemia. Fromdays 3 through 10, spleens on gross examinationwere normal to mildly enlarged; histologically,they showed congestion and neutrophils in thesinusoids of the red pulp.

Histologic pneumonic lesions were also firstobserved 3 days after animals inhaled 106 cellsof strain 425. Lesions were of the same progressivetypes but involved much more organ parenchymaand persisted longer than in monkeys given104 cells; several animals inhaling the higherdose still showed moderate pulmonary lesions60 days after exposure. Grossly, reddening wasobserved on day 3 and multiple areas of con-solidation and considerable pleural effusion wereseen from days 6 to 35. Pleural adhesions wereobserved through the 120th day. Ratios of lung

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weights to body weights (Fig. 5) show the in-crease in lung weight due to inflammatory infil-trate that reflects the relative degree of pulmonarydisease following inhalation of 104 or 106 orga-nisms. Pathologic changes in the tracheobronchiallymph nodes of monkeys receiving 106 cells wereseen from the 3rd through 15th day and weresimilar to those of animals inhaling 104 cells butwere much more extensive and also includedmoderately severe lymphocytic depletion andsubcapsular hemorrhage. Hepatic changes seenbetween the 3rd and 35th day were similar incharacter to those observed in animals given the104-organism dose, but the inflammatory cellinfiltrates were much more extensive. Spleenswere moderately enlarged and histologicallyshowed extensive congestion, neutrophil or mixedinflammatory cells in the red pulp, and hyalinechanges in the white pulp from days 3 through 35.

Roentgenographically, the lungs of animalsexposed to 104 or 106 cells and serially necropsiedwere generally normal through the third day. Ingeneral, the maximal changes were dose-related,occurred from days 6 to 15, and consisted ofdiffuse infiltrates up to 1 cm in diameter through-out both lungs to coalescence of infiltrates andlobar consolidation. Resolution was nearlycomplete by day 35, and no pulmonary abnor-malities were detected at 120 days.

Severity of lung lesions observed directly andby X ray paralleled the increase in the bacterialpopulation in the lungs and indicated maximalseverity of the disease from approximately day 5or 6 through days 10 to 15 after exposure re-gardless of dose. CRP and ESR also indicatedsimilar periods of maximal severity.

DISCUSSIONThe inability of F. tularensis strain 425, strain

Vt 68, or Soviet strain 503 to ferment glycerolor kill domestic rabbits inoculated subcutane-ously with 106 organism, is characteristic of type-B strains in man (11). These properties contrastsharply with those of strain SCHU S4 that is typeA for man.

In the present study, the monkey was evaluatedas a model for type-B human tularemia. Theseanimals were uniformly infected with strain 425;incubation periods were shorter, and the illnesswas longer and more severe with increasing in-haled numbers of organisms. Animals inhalinggraded doses of 10 through 103 cells usuallydeveloped mild to moderate acute illness andrecovered. Those given 104 through 106 organismsincurred a moderate to severe illness with 18%mortality at the highest dose. Throughout alldoses, the incubation period and duration ofacute illness were generally 3 to 7 days and 1 to3 weeks, respectively.

The 72 human tularemia cases reported in theVermont outbreak (3, 26) showed a wide spec-trum of disease ranging from severe illness in afew to inapparent infection in 24%o. The probable6-day median incubation period and 14-daymedian duration of illness were both within theranges observed in the present study. No deathsoccurred during the Vermont outbreak. Thehuman mortality from untreated tularemia inEurope and Asia is reported as less than 1 %(8, 15, 16).In contrast, fully virulent strain SCHU S4 (type

A) has a respiratory median lethal dose (LD50)<50 organisms for rhesus monkeys; 200 inhaledorganisms routinely resulted in death (6). In thepresent study, an LD50 was not attained inmonkeys inhaling as many as 106 cells. Type-Atularemia in man usually results in severe illness(6, 9, 17, 20) regardless of the route of infection,and a 30% case fatality rate is estimated for theuntreated pneumonic form (17). Thus the type-Aand type-B infections in rhesus monkeys ex-hibited the differences seen in the naturally oc-curring human cases, although monkeys are lessresistant than man. Information reported herefurther demonstrates the suitability of tularemiain monkeys as a laboratory model for man.Our data also support the use of rabbit virulenceand glycerol fermentation for classifying strainsin regard to virulence for man.The pattern of dissemination, multiplication,

and clearance of strain 425 in monkeys wasdose-related. Onset of regional and systemicinfection was shorter in animals given 106 cellsthan in those given 104 cells. Through the periodof regional infection, animals were asymptomaticand no tissue changes were observed; similarcoincidence of onset of clinical illness and sys-temic infection have been reported for guineapigs infected with Eurasian strain 503 (14).Mean maximal bacterial populations in thelungs of monkeys occurred on the sixth to seventhdays and were 1,000-fold higher than the inhaleddose. White et al. (24) demonstrated antitularensisgamma globulin in the tracheobronchial lymphnodes and spleen 5 days and in the lungs 7 daysafter aerogenic exposure of monkeys to F.tularensis live vaccine strain LVS. These datasuggest that this immune response may play arole in limiting strain 425 populations as recordedin the present study. Eigelsbach et al. (7) reportedthat monkeys inhaling 105 organisms of fullyvirulent type-A strain SCHU S4 exhibited a 10,000-fold increase in organisms in the lungs within2 days and died within 3 days after exposure. Inthe present investigation, animals did not dieuntil 6 to 7 days after exposure, and generallylungs contained 106 to 1010 organisms. These data

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PATHOGENESIS OF TULAREMIA IN MONKEYS

suggest that the rate of bacterial multiplicationand total population that occur before signifi-cant immune response develops largely reflect therespiratory virulence of F. tularensis strains.The extent of monkey tissue changes was

directly related to the strain 425 population in thetissues. The most significant tissue changes oc-curred in the lungs; severity of clinical illness wasrelated directly to pulmonary tissue changes. Incontrast to the self-limiting granulomatous lesionsresulting from type-B strain 425, Eigelsbachet al. (7) and White et al. (25) reported thatmonkeys exposed aerogenically to type-A strainSCHU S4 developed fulminating bronchopneu-monia and systemic infection with extensivenecrosis. Animals usually died before granulomasdeveloped. Fatal human cases exhibited acutenecrotic lesions primarily in the lungs, spleen,lymph nodes, and bone marrow (22).The moderate degree of pulmonary involve-

ment of monkeys observed on roentgenogramscorrelated directly with pathologic observationsduring the acute strain 425 infection, but residualpleural adhesions observed at necropsy afterclinical recovery were not detectable by X ray.Others (17) have reported that monkeys inhalingstrain SCHU S4 rapidly developed multiplebronchopneumonic infiltrates and multilobarconsolidation. Man inhaling type-A organismsdeveloped single or multiple pneumonic infiltrates,hilar adenopathy, pleural effusion, and lobarconsolidation; therapy resulting in completeroentgenographic resolution of lung lesions (17).The inhaled dose determined the maximal

bacterial growth in the lungs, which in turn influ-enced the severity of pneumonia and clinical ill-ness. The dependence of the length of the incuba-bation period and extent of pulmonary and othertissue changes on the inhaled bacterial dose isconsistent with the belief that the strain 425organisms act independently of each other andthat clinical and anatomic manifestations of theinfection and death occur when the bacterialburden attains a given level.

ACKNOWLEDGMENTS

We thank Joseph V. Jemski and Ellis J. Tonik for supervisingthe aerosol exposures; Jerome H. Krupp and Roger S. Boughtonfor their assistance with the necropsies; and Richard H. Pettit,Charles H. Remsberg and Ruth D. Herring for their technicalassistance. All are associated with the U.S. Army BiologicalDefense Research Center, Fort Detrick, Md. We also thank NelsonR. Blemly and Edward V. Stabb for interpreting roentgenograms.Both were formerly with the U.S. Army Medical Research InstitLteof Infectious Diseases, Fort Detrick, Md.

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25. White, J. D., J. R. Rooney, P. A. Prickett, E. B. Derrenbacker,C. W. Beard, and W. R. Griffith. 1964. Pathogenesis of ex-

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